Methods for improving analyte detection using photochemical reactions

ABSTRACT

An improved assay for detecting an analyte in a fluid sample includes a step of conducting a photochemical reaction, in which a substrate conversion catalyzed by a photosensitizer into a product of the photochemical reaction is temporary inhibited when the reaction mixture is irradiated with a light at a wavelength within a light absorption spectrum of the photosensitizer. The photosensitizer (or an enzyme to catalyze producing thereof) is attached to an entity having an affinity to the analyte, such entity is bound to the analyte prior to irradiation. To achieve temporary inhibition, certain additives are used such as ascorbic acid or its derivatives. The assay may increase the sensitivity of ELISA 20- to 100-fold.

CROSS-REFERENCE DATA

This is a continuation-in-part of the co-pending U.S. patent applicationSer. No. 14/071,287 filed Nov. 4, 2013 with the same title, incorporatedherein by reference in its entirety.

REFERENCE TO GOVERNMENT SPONSORED RESEARCH

This invention was made with government support in the form of a grantNo. AI055310 entitled “A new PCR-based method for HIV detection” and agrant No. AI083169 entitled “Ultrasensitive method for HIV p24 antigendetection”, both grants were awarded by the National Institute ofAllergy and Infectious Diseases (NIAID), National Institutes of Health(NIH). The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

This invention relates to methods for improving detection of variousphysiologically active substances in tested fluids

Immunoassays, hybridization, ligand-receptor and enzymatic assays arewidely used in biology and medicine for detection of viruses, bacteria,cancer markers, and for drug screening (Edberg, 1985; Catty, 1989).

In particular, immunoassay and hybridization assays are used fordetection of a number of pathogenic bacteria and viruses that can beconsidered possible biological warfare agents. Among them are an Anthrax(Bacillus anthracis), an Arenaviruse, a Clostridium botulinum toxin, aBrucella specie, a Burkholderia pseudomallei, a Chlamydia psittaci, aVibrio cholerae, an Ebola virus, a variola major, a Staphylococcalenterotoxin B, a Francisella tularensis, a Rickettsia prowazekii, aYersinia pestis, a Cryptosporidium parvum, and food- and water-bornebacteria, such as Salmonella sp., an Escherichia coli O157:H7, Shigella,Staphylococcus aureus, Campylobacter jejuni, Campylobacter coli,Listeria. monocytogenes and Bacillus cereus. They are included inbioterrorism pathogens list published by the Centers of Disease Controland Prevention (CDC). These agents are considered possible biologicalwarfare agents because they are resistant to environmental conditions,most of the human population is completely susceptible, and the diseasessome of them cause are severe with a high fatality rate. A largequantity of these organisms could easily be grown and preserved forseveral years. These agents are also environmental and food chain-safetythreats.

The effective testing of, for instance, pathogenic bacteria requiresmethods of analysis that meet a number of challenging criteria. Time andsensitivity of analysis are the most important limitations related tothe usefulness of microbiological testing. Bacterial detection methodshave to be rapid and very sensitive since the presence of even a singlepathogenic organism in the water, body or food may be an infectiousdose. At present, three groups of tests for food- and water-bornebacteria are, mainly, used: 1) culture methods, 2) immunoassay, and 3)polymerase chain reaction (PCR)-based assays. Culture methods arelaborious, time-consuming and very expensive. They require a minimum ofseveral days to perform. Polymerase chain reaction (PCR) method is basedon amplification of small quantities of genetic material to determinethe presence of bacteria. The PCR method is rather sensitive, butrequires pure samples and hours of processing and expertise in molecularbiology (Meng et al., 1996; Sperveslage et al., 1996). Immunoassaymethods, which yield a presumptive positive or negative screening resultin 24 h or less have also been developed. Typical sensitivities ofimmunoassays are approximately 10⁶ CFU/ml, although more sensitivevariants of immunoassay (with sensitivities of 10²-10⁵ CFU/ml) are beingdeveloped (Yu and Bruno, 1996; Mazenko et al., 1999).

Thus, there is a need in highly sensitive, rapid and reliable analyticalmethods for detection of water- and food-borne pathogens. In response tothis problem, considerable effort is now directed towards thedevelopment of methods that can rapidly detect low concentrations ofpathogens in water, food and clinical samples. For this purpose, anumber of instruments are being developed using various principles ofdetection, e.g. chromatography, infrared or fluorescence spectroscopy,bioluminescence, flow cytometry, impedimetry and many others (Ivnitskiet al., 1999). Despite these efforts, the only a few biosensors forbacterial detection are commercially available or are approachingcommercialization. The main reasons for this are the challenges tocreate biosensors with the necessary sensitivity and properties forreliable and effective use in routine applications. Since the presentinvention allows one to increase the sensitivity of the analyticalbiochemical methods significantly, it has a paramount value for a morereliable detection of potential biological warfare agents.

Very sensitive analytical biochemical methods are also of significantvalue for detection of infectious diseases. For example, in spite ofusing sensitive Enzyme-linked immunosorbent assays (ELISA) for thedetection of HIV, Hepatits B and C viruses in blood donors, the residualrisk of post-transfusion infection remains. The enzyme immunoassaytesting currently used for HIV and Hepatitis B testing produces manyfalse negative results. For example, deaths due to fulminantpost-transfusion hepatitis B have been reported although HBsAg-negativeblood on ELISA was transfused (Arababadi et al., 2011). The amount ofHBsAg contained in the peripheral blood of these HBsAg-negative HBVcarriers is so small that the currently employed methods are inadequate(Sawke and Sawke, 2010).

Other physiologically active substances that require high sensitivitymethods to be detected are pathogenic bacteria, viruses, cancerbiomarkers such as Clostridium difficile toxin A, a Clostridiumdifficile toxin B, a rotavirus, a p50 recombinant protein NFkB p50homodimer, an RNA, a DNA, an mRNA, an cDNA, and a prostatic specificantigen (PSA).

Formats of Analytical Biochemical Assays

There are a number of various formats of immuno- and hybridization assayand ligand-receptor assays that are used in clinical and researchpractice. They are based on the use of either antigen-antibody,ligand-receptor or oligonucleotides hybridization reactions fordetection of the analyte to be detected, and use of labels (markers) ofdifferent origin: photochemical, radioactive, enzymatic, fluorescent,chemiluminescent and others.

1. Assays Based on the Use of Photochemical Labels

It has been reported that certain dyes, for instance derivatives offluorescein, rhodamine, erythrosine, eosin, methylene blue, Bengal rose,porphyrin, phthalocyanine and many others were used as photochemicallabels in various analytical biochemical assays. These dyes possess ahigh yield to their triplet state, and therefore, can be used asphotosensitizers (photocatalysts) of the photochemical reactions. Undercertain conditions photochemical reaction between such dye and selectedsubstrate occurs and takes place only under irradiation with certainrange of light wavelengths.

Photochemical Reactions and Photooxidation.

Chemical reactions caused by absorption of light are defined asphotochemical reactions. Photoexcitation is the first step in aphotochemical process in which the photon can be absorbed directly bythe reactant or by a photosensitizer which absorbs the photon andtransfers the energy to the reactant. There are several types ofphotochemical reactions. One such type is photosensitized oxidationimplicated in numerous processes in vivo and in vitro. Many of theseoxidation processes can occur in the dark; however, light can accelerateoxidation due to the photochemical generation of free radicals (type Iphotosensitized oxidation) or singlet oxygen (type II photosensitizedoxidation). Strongly light-absorbing organic dye molecules such as RoseBengal, erythrosine, eosin, methylene blue, porphyrines andphthalocyanines are typical examples of photosensitizers that canparticipate in Types I and II photochemical reactions. In Type IIphotochemical reaction, produced singlet oxygen reacts with many organiccompounds including aromatics, vitamins, steroids, fatty acids,aminoacids, proteins, nucleic acids, and synthetic polymers (Timoshenko,2009). Photosensitized oxidation involving singlet oxygen is implicatedin analytical assays, phototherapy of cancer, photocarcinogenesis,photodynamic inactivation of viruses and cells, and in photodegradationof organic compounds (Schmidt, 2006b; Schmidt, 2006a).

Various formats of analytical assays using photosensitizers as labelsfor determination of physiologically active substances (analytes) havebeen described in patent and scientific literature. In references(Motsenbocker et al., 1993a; Motsenbocker et al., 1993b) derivatives ofmethylene blue were used as labels in Enzyme-Linked Immunosorbent Assay(ELISA)-type assays. In this assay, a methylene blue dye derivative wassynthesized and covalently attached to detection antibody. Uponirradiation of the solution containing luminol by pulsed red light, aphotosensitive dye is excited and plays a role of photosensitizer(photocatalyst) of luminol oxidation which results in generation of bluelight. An alpha-fetoprotein immunoassay based on this principle wasdeveloped having a detection limit of 17 pg.

It has also been reported that fluorescein, rodamine and eosin can beutilized as photochemical antibody labels in immunohistochemistry. Itwas shown that DAB can be photooxidized by these dyes in immunolabeledcultured cells (Sandell and Masland, 1988). In this case, thefluorochrome which is a label for the antibody bound to the cell, isutilized as a photosensitizer in the photochemical reaction of DABoxidation. Since the production of reactive oxygen species by thefluorophore has been implicated in the photooxidation reaction (Sandelland Masland, 1988), the experiment made use of test fluorescentcompounds that were more potent generators of singlet oxygen. Many ofthe compounds currently used for immunofluorescent and tyramine-basedlabeling, such as fluorescein and rhodamine were chosen because of theirhigh fluorescence quantum yields, and have comparatively low yields ofsinglet oxygen (¹O₂). Eosin, a brominated derivative of fluorescein, hasa singlet oxygen quantum yield (0.57) approximately 19 times greaterthan fluorescein (Gandin et al., 1983), while still possessing moderatefluorescence (˜20% as bright as fluorescein) (Fleming et al., 1977).Other fluorescein derivatives such as erythrosine and Rose Bengal arealso known to be effective photosensitizers and singlet oxygengenerators, and can be used as labels in immunohistochemistry.

Two homogeneous (not requiring physical separation of labeled andunlabeled reagents) immunoassay techniques based on the use ofphotogeneration of singlet oxygen have been independently developed. Thefirst method is based on (a) photooxidation by singlet oxygen (¹O₂) of afluorescent substrate (1,3-diphenylisobenzofuran, DPBF) embedded inunilamellar vesicles on the surface of which antibody to the analyteantigen is covalently attached (DPBF-immunoliposomes); (b) generation ofsinglet oxygen, upon illumination, by a chromophore (erythrosine)covalently attached to an antibody (Ab*) or antigen (Ag*); (c) formationof a “sandwich”- or “competition”-type complex whereupon the singletoxygen-generating chromophore conjugate (Ab* or Ag*) andimmunoliposome-embedded DPBF are brought into close proximity (Bystryaket al., 1995; Bystryak, 1998). Competition- and sandwich-type modelassay systems for the detection of protein antigens and viruses weredeveloped.

In the second method, the Luminescent Oxygen Channeling Immunoassay(LOCI) (Ullman et al., 1994), singlet oxygen is generated by aphotosensitizer and an antenna dye that are dissolved in one of theparticles. Singlet oxygen molecule diffuses to the second particle andinitiates a chemiluminescent reaction of an olefin that is dissolved init. The technique permits real-time measurement of particle bindingkinetics when analyte is present in the solution. By usingantibody-coated particles, a homogeneous immunoassay capable ofdetecting approximate to 4 amol of thyroid-stimulating hormone in 12 minwas demonstrated.

AlphaScreen/AlphaLISA assays developed based upon an oxygen channelingtechnology, LOCI are bead based proximity assays when the donor, whichcontains phthalocyanine, is laser excited and ambient oxygen isconverted to singlet oxygen. This is a highly amplified reaction sinceapproximately 60,000 singlet oxygen molecules can be generated andtravel at least 200 nm in aqueous solution before decay. Consequently,if the Donor and Acceptor beads are within that proximity, energytransfer occurs. Singlet oxygen reacts with chemicals (substrates) inthe Acceptor beads to produce a luminescent response (Eglen et al.,2008). AlphaScreen/AlphaLISA assays are immunoassays as well as highthroughput drug screening assays involving antigen-antibody,oligonucleotide hybridization, biotin-streptavidin, ligand-receptorbinding reactions and enzymatic reactions.

2. Assays Using Enzymes as Labels

Since the introduction, in 1966, of enzymes as markers for the labelingof antigens and antibodies (Avrameas and Uriel, 1966; Nakane and Pierce,1966), immunoenzymatic techniques have been considerably developed anddiversified. These techniques are now routinely used for detection ofphysiologically active substances in body fluids, localization ofantigens or antibodies on tissues, detection of antigens or antibodiesimmobilized on various solid phases, as well as for the titration ofantibodies, and for the precise measurement of antigens. Antigens and/orantibodies are localized, detected and/or titrated by means of variousheterogeneous procedures.

In general, heterogeneous immunoenzymatic procedures are based on theuse and detection of enzyme-antibody or enzyme-antigen conjugates,prepared according to several established protocols. Sometimes, antibodyor antigen is coupled to enzyme through biotin-streptavidin bond, whichis antibody (or antigen)-biotin-streptavidin-enzyme conjugate is used.The enzyme is detected using chromogenic, fluorogenic orchemiluminescent enzyme substrates by detecting a corresponding signalobtained from the product of the enzymatic reaction.

For example, in conventional Enzyme Linked Immuno-Sorbent Assay (ELISA),antibody against analyte to be detected is attached to solid phase,particularly, polystyrene microtiter plate. Then, body fluid withunknown concentration of the analyte and enzyme-labeled antibody toanalyte are added. This results in formation ofantibody-analyte-enzyme-labeled antibody complex on the surface of thesolid phase. After incubation, the unbound analyte and enzyme-labeledantibody is removed from the solution by rinsing. Then, a substratesolution is added to the test tube or well, and the amount of the boundenzyme-conjugated antibody and, consequently, the concentration of theanalyte is determined by detecting color, fluorescence or luminescencesignals of the liquid phase in the final reaction mixture.

Other than ELISA methods that use enzyme as a label includehybridization assays, Immunohistochemistry (IHC) and in situhybridization (ISH) assays, blotting analysis, immunochromatography andother methods. Hybridization and in situ hybridization (ISH) assays arebased on oligonucleotides binding whereas Immunohistochemistry (IHC),blotting analysis and immunochromatography methods are based onantigen-antibody reactions in the same way as ELISA.

However, in many cases, the sensitivity of these assays is inadequate.Therefore, there is a need in development of more sensitive assays. Inorder to detect constituents (analytes) present in small amounts, it isnecessary to devise procedures capable of strongly amplifying the signaland/or increasing of signal-to-noise ratio, which results in increasingof the analytical sensitivity of the assay. Since all heterogeneousimmunoenzymatic techniques involve, in their final step, the detectionof an enzyme associated with solid phase, essentially two approacheshave been developed to obtain such enzymatic signal amplification. Thefirst includes procedures leading to a high accumulation of enzymelabels associated with the solid phase. The second consists ofprocedures that make use of enzyme substrate derivatives, which giverise to reaction products detectable in minute amounts. The presentinvention relates to the second approach, which is also applicable tosome assays using photosensitized photochemical reactions.

A type of amplification technique that can be employed forenzyme-mediated assays utilizes light or photonic energy to increase thesensitivity of the assay. This technique is disclosed in U.S. Pat. No.5,776,703. It is widely known that some chemical reactions arephotosensitive dependent upon the quantum chemical structure and otherproperties of the reactants. The rate of a photosensitive reaction ismuch higher if the reaction mixture is illuminated by an intense lightof a specific wavelength compared to a similar reaction taking place inthe absence of such light.

The technique described in the '703 patent includes the binding of anantibody to a suspected antigen, wherein the antibody is labeled with anenzyme such as HRP and added to a biological liquid, for example bloodor serum. A portion of the HRP-labeled antibodies binds with theantibodies that are specific to the antigen and existing already in thebiological liquid to form an [antibody]-[antigen]-[HRP labeled antibody]complex. Subsequently, after an incubation time, the HRP-labeledantibody which did not bind to the Ab-Ag complex is removed from thesolution by rinsing or washing. Then, a substrate solution containingH₂O₂ and OPD (orthophenylenediamine) is added to the test tube and theOPD is oxidized with HRP acting as an oxidizing catalyst. The oxidationproduct of HRP-catalyzed reaction of OPD oxidation is diaminophenazine(DAP). DAP is a colored substance, and the optical density of thesolution containing DAP can be read with the aid of a spectrophotometeror microplate reader. At this point in the assay a stopping solution,such as sulfuric acid, is used in order to stop the various chemicalreactions from proceeding and producing reaction products that mayinterfere with an accurate measurement. The final signal is proportionalto the concentration of HRP bound to antibodies and therefore to theadded antibodies forming the Ab-Ag-(Ab-HRP) complex.

The '703 patent further discloses that the procedure performed to thispoint can be enhanced by the application of intense light at thewavelength of 400 to 500 nm before adding a stop solution containingacid. Prior to a spectrophotometer (or fluorometer) reading, the testtube is illuminated by an intense source of light of a wavelength in theabove range. The DAP obtained in the first stage of the reaction is aphotosensitizer for the following photochemical reaction of OPDoxidation, and together with the light photons serve as new catalyzingagents for further production of DAP. Thus a two stage process takesplace:

The resulting optical density or fluorescence of the sample is measuredby a spectrophotometer or fluorometer, respectively, and the resultsobtained by the '703 patent are an improvement over the prior art.However, this method suffers from the limited sensitivity due to thefact that both useful and background (noise) signals increase within aphotochemical step (1.2). This severely restricts the utility of themethod. It should be emphasized that other binding agents and pairs suchas oligonucleotides, oligonucleotide-protein,biotin-avidin(streptavidin) and protein-protein can be used in such kindof assays.

An attempt of modification of the technique disclosed in U.S. Pat. No.5,776,703 was done in US PATENT APPLICATION PUBLICATION No.2002/0110842, which is incorporated herein by reference in its entirety.The '842 application describes adding of some detergents to the OPDsubstrate solution to increase the sensitivity of the assay including aphotochemical amplification step. The best mode of practicing the '842invention included the commercially available detergent Triton X-100 asan additive at a concentration in the range of 10% in aphosphate/citrate buffer with a pH of approximately 5.0. Triton X-100has a chemical formula of C₁₄H₂₂O(C₂H₄O)n, where the average number ofethylene oxide units per molecule, n, ranges from 9 to 10.

Also disclosed is the use of Tween-20, another commercially availabledetergent, as an additive. However, the addition of detergents to theOPD substrate solution results in increase of the rate of thephotochemical reaction (1.2) only; the sensitivity of the method was notimproved as compared to the method described in the '703 patent.

Although the methods described in '703 patent and '842 application haveincreased the sensitivities of assays versus prior art methods, it isstill desirable to have an even more sensitive technique. In addition tosensitivity, it would be beneficial to have an immunoassay method thatfurther improved the signal-to-noise (S/N) ratio, which wouldsubstantially increase the effective range of the known methods. Themethods of the '703 patent and the '842 application are adverselyaffected to a large degree by background noise. In addition, thesemethods are highly dependent upon a number of factors including: (1) thewavelength of the light used to catalyze the reaction; (2) the intensityof the light (the number of photons used to catalyze the reaction); and,(3) the illumination exposure (intensity x time).

It is therefore desirable to have an assay of detecting an analyte in asample with increased sensitivity compared to known assays. It is alsodesirable to have an assay for detecting an analyte in a sample with animproved S/N ratio compared to known assays. It is also desirable tohave an assay for detecting an analyte in a sample that can be used withexisting ELISA and other assays methodology.

SUMMARY OF THE INVENTION

A photochemical method of the present invention (PMI) includes carryingout an analytical assay for detection of the analyte and utilizingphotochemical reactions in which certain additives are used to increasethe signal-to-noise ratio, sensitivity and rate of the analytical assay.The assay may be commercialized as a kit comprising novel solutions ofthe additives, the addition of which results in increased sensitivity ofthe conventional testing methods and/or rates of the photochemicalreaction.

It is therefore an object of the invention to provide an assay fordetecting an analyte in a sample, wherein the assay utilizes aphotochemical reaction. The addition of the reagents described in thisinvention has increased sensitivity compared to known conventionalassays and other methods utilizing photochemical reaction.

It is another object of the invention to provide an improved assay fordetecting an analyte in a sample with an improved signal-to-noise ratio(S/N ratio) and sensitivity as compared to known assays utilizingphotochemical reactions.

These and other objects are obtained by providing an assay for thedetermination of an analyte in a fluid sample comprising a step ofconducting a photochemical reaction, in which a substrate conversion iscatalyzed by a photosensitizer into a product of the photochemicalreaction. This reaction is temporary inhibited when the reaction mixtureis irradiated with a light at a wavelength within a light absorptionspectrum of the photosensitizer. The photosensitizer or an enzyme tocatalyze producing of the photosensitizer is attached to an entityhaving an affinity to the analyte. The entity is bound to the analyteprior to irradiation with the light.

It is yet another object of the invention to provide an improved assayfor detecting an analyte in a sample, which may be compatible withexisting ELISA methodology.

Yet another object of this invention is to provide improved assays todetect HIV p24 antigen, Hepatitis B and C antigens and antibodies,prostate specific antigen, E. coli and Listeria bacteria and otherpossible biological warfare agents and other physiologically activecompounds and analytes.

The method of the invention includes the steps of binding to the analyteof an entity having an affinity to the analyte. This entity may belabeled with a photosensitizer or an enzyme to catalyze producing of thephotosensitizer. Some additive or additives such as ascorbic acid or itscertain derivatives may be added to the solution before performing thefollowing photochemical reaction. Thus, the sample is irradiated withphotonic energy, whereby the additives and the photonic energy providecatalysis for further production of the products of the photochemicalreaction. The absorbance (OD), fluorescence, chemiluminescence orelectrochemical parameters of the product of the photochemical reactionmay be then measured to determine the concentration of this product,and, thereby, the analyte concentration in the fluid may be determined.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A through 1D describe effect of various additives contained inreagent solutions on the calibration curves and kinetics of DAP-mediatedphotochemical reaction of OPD oxidation as follows:

FIG. 1A shows calibration curves obtained at various times ofillumination for detection of the DAP primer added to the OPD substratesolution in the absence of the reagent solution (no additives);

FIG. 1B shows kinetics of the DAP-mediated reaction of OPD oxidation inthe absence and presence of the reagent solution containing 250 μMascorbic acid;

FIG. 1C shows calibration curves obtained at various times ofillumination for detection of the DAP primer added to the OPD substratesolution in the presence of the reagent solution containing 250 μMascorbic acid;

FIG. 1D shows calibration curves obtained at various times ofillumination for detection of the DAP primer added to the OPD substratesolution in the presence of the reagent solution containing 500 μMascorbic acid and 1.5% Tween 20.

FIGS. 2A through 2D show calibration curves for determination of HRPconjugate in the buffer solution at various times of illumination in theabsence (FIG. 2A) and presence of the reagent solutions containing 250μM ascorbic acid (FIG. 2B), 250 μM ascorbic acid and 1.5% Tween 20 (FIG.2C) and 500 μM ascorbic acid and 1.5% Tween 20 (FIG. 2D).

FIGS. 3A through 3C show calibration curves for determination of HRPconjugate in the buffer solution in the presence of the reagentsolutions containing 125 mM ascorbic acid 6-palmitate and 1.5% Tween-20(FIG. 3A), 250 mM isoascorbic acid+1.5% Tween-20 (FIG. 3B), and 18.6%glycerol and 1.5% Tween-20 (FIG. 3C).

FIG. 4 shows calibration curves for determination of HIV-1 p24 antigenusing conventional ELISA (0 min illumination) and ELISA+PMI at varioustimes of illumination.

FIGS. 5A through 5D illustrate calibration curves for determination ofHIV-1 p24 antigen using conventional non-ICD ELISA (PerkinElmer) (FIG.5A), non-ICD ELISA+PMI (FIG. 5B), heat-mediated ICD ELISA (FIG. 5C), andheat-mediated ICD ELISA+PMI (FIG. 5D).

FIGS. 6A through 6D show calibration curves for determination of NFκBp50 homodimer using conventional ELISA (ActiveMotif) and p50 recombinantprotein (FIG. 6A), ELISA+PMI p50 recombinant protein (FIG. 6B),conventional ELISA and nuclear extracts (FIG. 6C), and ELISA+PMI andnuclear extracts (FIG. 6D).

FIGS. 7A through 7E show determination of HIV-1 viral load usingAMPLICOR HIV MONITOR Test, version.1.5 (Roche)+PMI using less powerfulillumination device as follows:

FIG. 7A shows dependences of ODs for low positive control (at startingdilution 1:64) on dilution factor at various times of illumination;

FIG. 7B shows dependences of ODs for Quantitation standard (at startingdilution 1:5) on dilution factor at various times of illumination;

FIG. 7C shows the signals (ODs) obtained for sample PS at startingdilution 1:200 and Quantitation Standard at dilutions 1:10 and 1:50 atdifferent times of illumination as a function of a dilution factor;

FIG. 7D shows HIV-1 loads calculated using a modified protocol; and

FIG. 7E shows the average HIV-1 loads calculated using original andmodified protocols.

FIGS. 8A through 8C show using of OPD as an insoluble HRP substrate inmembrane- and cell-based assay as follows:

FIG. 8A shows a comparison of HRP substrates: Signals obtained forHRP-conjugated antibody spotted on nitrocellulose membrane (GEHealthcare) at decreasing concentrations (dilutions of its stocksolution are shown on the right) and immersed into the indicatedsubstrate solutions;

FIG. 8B shows the signals obtained for HRP-conjugated antibody spottedon nitrocellulose (strips 1 and 3) and Nylon membranes (strips 2 and 4)without blocking (strips 1 and 2) and with blocking for 2 hours at roomtemperature (strips 3 and 4) and immersed into OPD substrate solution;and

FIG. 8C shows signals obtained for HRP-conjugated antibody spotted onNylon membrane at decreasing concentrations (dilutions of its stocksolution are shown on the right) without (strip 1, TMB and strip 2, OPD)and with PMI (OPD and the reagent solution containing 250 μM ascorbicacid 1.5% Tween 20).

DEFINITIONS

“Reagent solution” or “Inhibitors” or “Retarders” means a solution orother physical form (solid, liquid, suspension, tablet) comprising oneor more novel additives described in this disclosure: antioxidants,alcohols, detergents (surfactants), sugars, polymers, organic andinorganic reducers, and scavengers of reactive oxygen species (ROS).

“Antioxidant” means a molecule capable of slowing or preventing theoxidation of other molecules. Non-limiting examples of antioxidantsinclude ascorbic and iso-ascorbic acids and their derivatives (suchascorbates and iso-ascorbates, ascorbic acid palmitate, stearate, andthe like), tocopherols and trienols (alpha-, beta-, gamma- anddelta-tocopherols and trienols), their derivatives and synthetic solubletocopherol analogs similar to Trolox, flavanoids (myricetin, quercetin,rutin, kaempferol, and the like), antioxidant phenolic compounds otherthan flavanoids (BHA, BHT, TBHQ, riboflavin, tert-butyl phenol, gallicacid, caffeic acid, and similar), thiols and hydrosulfites(mercaptoethanol, L-cystein, dithiothreitol, dithionite,dithioerythritol and the like), carotenoids (xanthopylls and carotenes),melatonin, uric acid and derivatives, ubiquinones and quinones,lactates, NADH and its analogs, calcium antagonists, vitamin B6,aspirin, steroids pyrrolopyrimidines, ebselen, metallothioneins,metalloporphyrins, fullerenes and antioxidant mixtures.

“Alcohol” means a compound of the formula C_(n)H_(2n+1)(OH)_(x), whereinn>/=2, x=/>1. Examples include but are not limited to methanol, ethanol,glycerol and ethyleneglycol (HO—CH₂—CH₂—OH) and their polymer analogs.

“Surfactant” means a substance that lowers the surface tension of aliquid. They may be ionic or non ionic. Non-limiting examples ofsurfactants include:

“Ionic surfactant” may include the following by way of an example:

-   -   1. Anionic (based on sulfate, sulfonate or carboxylate anions)        -   a) Perfluorooctanoate (PFOA or PFO)        -   b) Perfluorooctanesulfonate (PFOS)        -   c) Sodium dodecyl sulfate (SDS), ammonium lauryl sulfate,            and other alkyl sulfate salts        -   d) Sodium laureth sulfate, also known as sodium lauryl ether            sulfate (SLES)        -   e) Alkyl benzene sulfonate        -   f) Soaps, or fatty acid salts    -   2. Cationic (based on quaternary ammonium cations)        -   a) Cetyl trimethylammonium bromide (CTAB) a.k.a. hexadecyl            trimethyl ammonium bromide, and other alkyltrimethylammonium            salts        -   b) Cetylpyridinium chloride (CPC)        -   c) Polyethoxylated tallow amine (POEA)        -   d) Benzalkonium chloride (BAC)        -   e) Benzethonium chloride (BZT)    -   3. Zwitterionic (amphoteric)        -   a) Dodecyl betaine        -   b) Cocamidopropyl betaine        -   c) Coco ampho glycinate

“Nonionic surfactant” may include the following by way of an example:

-   -   1. Alkyl poly(ethylene oxide)    -   2. Alkylphenol poly(ethylene oxide)    -   3. Copolymers of poly(ethylene oxide) and poly(propylene oxide)        (commercially called Poloxamers or Poloxamines)    -   4. Alkyl polyglucosides, including:        -   a) Octyl glucoside        -   b) Decyl maltoside    -   5. Fatty alcohols        -   a) Cetyl alcohol        -   b) Oleyl alcohol    -   6. Cocamide MEA, cocamide DEA    -   7. Polysorbates: Tween 20, Tween 80        -   a) Dodecyl dimethylamine oxide

“Sugar” include the following examples: monosacharides (erythrose,arabinose, ribose, glucose, mannose, xylose, fructose, galactose, andthe like), disaccharides (sucrose, maltose, cellobiose, lactose, and thelike), oligosaccharides (raffinose, stachyose, cyclodextrins, and thelike).

Non-limiting examples of “polymers” include polysaccharides (amylose,amylopectins, glycogen, cellulose, their derivatives, and the like), andpolyethylene glycols.

“Scavengers of reactive oxygen species (ROS)” include but are notlimited to non-enzymatic scavengers such as antioxidants withhigh-reducing potentials and enzymatic scavengers such as superoxidedismutase (SOD), superoxide reductases, catalase, thioredoxin reductase,alkenal/one oxidoreductase, and peroxiredoxins.

“PA” or “PAM” refers to Photo Amplification or PhotoamplificationMethods disclosed in U.S. Pat. No. 5,776,703 and US patent applicationpublication No. 2002/0110842.

“PMI” refers to the Photochemical Method of the present Invention.

DETAILED DESCRIPTION OF THE INVENTION

An improved assay of the present invention for detecting an analyte in afluid sample comprises a step of binding an entity labeled with aphotosensitizer (or an enzyme to catalyze producing a photosensitizer)to the analyte. A critical step of the process is adding a reagentsolution with an additive to cause a temporary inhibition of aconversion of a substrate added to a mixture of the entity and theanalyte into a product of a photochemical reaction. Once the additive isdepleted, this conversion is accelerated while a mixture of the entity,the analyte and the substrate is irradiated with a light at a wavelengthwithin a light absorption spectrum of the photosensitizer.

In embodiments, the assay of the invention may be conducted by followingthese steps:

-   -   a) binding the analyte to a first entity having an affinity to        the analyte, this entity labeled with a photosensitizer or an        enzyme to catalyze producing of the photosensitizer,    -   b) adding to a mixture of the analyte and the entity a substrate        for a photochemical reaction, which substrate is capable to be        converted to a product of the photochemical reaction when the        mixture of the entity, the analyte and the substrate is        irradiated with a light at a wavelength within a light        absorption spectrum of the photosensitizer,    -   c) adding a reagent solution containing an additive described by        formula (1)

-   -   wherein the additive is an ascorbic acid or a derivative thereof        obtained by at least one substitution at position 2, position 3,        position 5, position 6 or any combinations thereof with at least        one group of atoms selected from a group consisting of an anion        group of atoms, an aliphatic group of atoms not exceeding 16        carbon atoms, and an aromatic group of atoms not exceeding 16        carbon atoms;    -   d) conducting the photochemical reaction by irradiating the        mixture of step (c) with the light, and    -   e) detecting the analyte by measuring an optical signal        corresponding to the amount of the product of the photochemical        reaction.

In embodiments, the entity having an affinity to the analyte may be anantibody, an antigen, a ligand or a receptor. Dyes may be attached as alabel to such entity. Certain dyes, for instance derivatives offluorescein and rhodamine may be used as labels or markers of antibodiesand antigens. In one embodiment of the invention, the photosensitizedmaterials used as labels may include dyes with a significant yield totheir excited triplet state. Exemplary dyes may include derivatives ofphenazine, fluorescein, eosin, erythrosine, phthalocyanine, porphyrin,aminolevulinic acid, chlorine, purpurin, methylene blue, and Bengalrose.

In embodiments, a photochemical reaction between such dye and asubstrate may take place only under irradiation with light of definedwavelength. The products of such photosensitized reactions may becolored, fluorescent, chemiluminescent or electrochemical materialswhich may be detected and measured using such known instruments andspectrophotometer (optical density reader), fluorometer,chemiluminometer or tools to measure electrical current parameters,respectively.

The principal difference (and advantage) of the assay of the presentinvention as compared to other known photochemical diagnostic proceduresis the use of additives to the substrate solution to temporary inhibitthe photochemical reaction—until such additives are depleted. Use ofsuch additives leads to acceleration of the photochemical reaction oncethe inhibition is over—leading to an overall increase of thesignal-to-noise ratio and, hence, analytical sensitivity of the assay.

In embodiments, the additives may include an ascorbic acid or a certainderivative thereof, one or a combination of antioxidants, alcohols,surfactants, sugars, organic and inorganic reducers, and scavengers ofreactive oxygen species (ROS) as specified above.

In other embodiments of the invention, the substrates of thephotosensitized chemical reaction may include derivatives of OPD,Diaminobenzidine (DAB), olefin, luminol, dioxetane, and benzofurane.

In embodiments, examples of a photosensitizer include a phenazine, aphenazine derivative, a 2,3-diamino-phenazine, a benzidine, a benzidinederivative, an eosin, an eosin derivative, an erythrosine, anerythrosine derivative, a toluidine blue, a crystal violet, amerocyanine 540, Rose Bengal, a methylene blue, a porphyrine, ahematoporphyrin, a porphyrine derivative, a phthalocyanine, aphthalocyanine derivative, an aluminum phthalocyanine tetrasulfonate(AlPCS) derivative, a riboflavin, and a quantum dot.

In embodiments, the substrate or the photosensitizer may be embedded inmicroparticles, nanoparticles or liposomes. Some or all steps of theprocess may be carried out in a single mixture of all solutions withoutphysical separation of bound and unbound fractions of reagents.

The method is further described as it applies to a commerciallyavailable ELISA tests, which is meant only to be illustrative of themethod and not to limit the scope of the claims that follow. One skilledin the art would recognize the utility of the present invention in otherassays utilizing photochemical reactions. The description of other thanELISA methods is also provided.

The methodology of the assay of the invention may closely follow theprotocols of commercially available immunoassay and hybridizationassays. It is well understood by those skilled in the art that thepresent invention will have numerous applications in the field ofimmunoassay, ligand-receptor, hybridization assays and high throughputdrug screening and the scope of the invention will become more apparentthrough the following examples.

In one embodiment, described is a method for increasing ELISAsensitivity by utilizing a photochemical amplification step. It consistsof two critical steps. During the first step, a conventional horseradishperoxidase (HRP)-mediated assay may be used. An enzymatic label such asHRP may be used to catalyze the conversion of oxidation of a commonchromogenic HRP substrate such as orthophenylenediamine (OPD) into aproduct of a photochemical reaction, a photosensitizer2,3-diaminophenazine (DAP) (reaction 2).

During the second critical step, the reagent solution containing theadditives described above is added. The HRP substrate solutioncontaining the product of enzymatic reaction, 2,3-diaminophenazine(DAP), may be irradiated by light within a predefined range ofwavelengths. (reaction 3). In this example, the preferred range ofwavelengths is from about 400 nm to 500 nm. The term “about” is usedherein and throughout the specification to describe +/−30% deviationfrom the cited parameter. Irradiation of the samples with such lightleads to DAP-catalyzed drastic increase in DAP concentration(autocatalytic photochemical reaction). This reaction is delayed untilthe additives are depleted, leading to acceleration thereof afterwards.

Thus a two stage process takes place:

The assay of the present invention including ELISA and the photochemicalamplification as described may be called ELISA+PMI.

While the previous assays (described in the '703 patent and the '842application) were shown to increase the sensitivity up to 8-fold, thepresent invention is shown to achieve as much as a 100-fold sensitivityincrease. Furthermore, the use of the PMI step as described here allowsincreasing of the signal-to-noise ratio and decreasing of the limit ofdetection (LOD) of clinically significant analytes such as Clostridiumdifficile toxin A, a Clostridium difficile toxin B, HIV-1 p 24 antigen,Prostate Specific Antigen (PSA) and Hepatitis B surface antigen (HBsAg)and other physiologically active substances 20- to 100-fold, as comparedto conventional ELISA.

In ELISA-type assays, as an initial step, at least one interactivematerial, typically an antibody, may be retained or bound to a supportphase, which may comprise a plate, a body of beads or other particulatematter, tubes, rings, a porous matrix (such as those used in WesternBlot techniques), or other materials of various design known to thoseskilled in the art.

Next, a sample of the biological fluid to be tested may be added to theplate. Examples of the fluid samples may include blood, serum, plasma,different body fluids, food extract, environmental samples, cellculture, etc. If the biological fluid contains the target analyte(antigen) then an antibody-antigen complex is formed.

Next, an interactive material (such as an antibody to the specificantigen) may be added to the plate. This second antibody may be labeledwith biotin, and then enzyme conjugate of streptavidin may be used, orwith various enzymes that are well-known in the art. Of particularrelevance to the present invention, the enzyme used becomes part of acomplex that reacts with a substrate to produce a product which isdetectable using colorimetry, fluorometry or chemiluminescence. As anexample, the PerkinElmer HIV-1 p24 kit uses a streptavidin proteinlabeled with HRP which reacts with OPD to form DAP, which produces ayellow color that is detectable by absorbance (optical density) readers.

In the conventional methods, a stopping agent would now be applied aftera suitable reaction time. As opposed to prior art, the assay of thepresent invention delays or in some case avoids entirely the use of thestop solution. After enzymatic reaction, a reagent solution containingadditives of the present invention is added. Light irradiation is thenapplied. The photochemical reaction is temporality inhibited by theadditive as described above. Once the additive is depleted, thephotochemical reaction of OPD oxidation is accelerated to produce theproduct, DAP.

The invention also describes methods for increasing the sensitivity ofthe assay including the photochemical reaction. The invention furtherdescribes a method for increasing the rate of the photochemical reactionby the addition of one or more novel reagent solutions described herein.Further, the invention comprises a method for increasing both thesensitivity of the assay including the photochemical reaction and therate of the photochemical reaction.

In embodiments, the peroxidase enzyme is horseradish peroxidase.Furthermore, the light irradiation may activate a peroxidase reactionproduct of OPD oxidation. The reaction product of oxidation may be2,3-diaminophenazine (DAP).

In order to increase the slope of the calibration curve, and therebylower the detection limit of determination of HRP, certain additives tothe OPD substrate solution before or after the enzymatic reaction stepmay be added. These substances restrain the photochemical formation ofDAP at low HRP concentrations and, hence, at small amounts of DAP,produced during enzymatic reaction. That is, the additives areinhibitors of the reaction of OPD photooxidation (reaction 3) and theyhave to be depleted after a certain period of time. The reaction (3)proceeds through the formation of either OPD intermediates, for exampleOPD-cation radical, or reactive oxygen species (ROS)-intermediates.Addition of inhibitors of the reaction (3) to the OPD substrate solutionmay result in the interaction of inhibitor and reactive intermediatecompounds. This leads to the restraining of the signal increase atinitial stages of the reaction when inhibitor is yet to be depleted. Aschematic presentation of the reactions between inhibitor X andintermediates is as follows:

-   -   where X refers to the inhibitor interacting with intermediate        compounds to give non- or less reactive X-derivative. Inhibitor        X may inhibit the photochemical reaction until the moment when        amount of X is enough to restrain the formation of DAP from        intermediate compounds. Since the addition of X leads to        decrease in the amount of intermediates produced during the        DAP-photosensitized reaction, the initial rate of this reaction        will depend on the concentrations of X and DAP. That is, the        lower DAP concentration, the lower initial rate of the        photochemical reaction. Therefore, the light screening or        quenching effects at higher DAP will be compensated by even more        pronounced effect of the decrease in the rate of this reaction        at lower DAP concentrations caused by the addition of the        compound X. By another words, X is an organic compound added to        temporally inhibit photooxidation of OPD. Depletion of X is DAP        concentration dependent, and this depletion is followed by the        increased reaction rate of OPD autocatalytic oxidation (that is,        K₂>K₁).

In embodiments, the reagent solution comprises inhibitors X (additives)which may include one or a combination of antioxidants, alcohols,surfactants, sugars, organic and inorganic reducers, and scavengers ofreactive oxygen species (ROS).

Antioxidants may be classified into several categories based on theiroccurrence, structure, mode of action, kinetics, and solubility. Thesecategories may include vitamins, hormones, steroids, saponines,carotenoids, enzymes, glutathione, minerals, phenolics, and lipidassociated chemicals. These are also classified as enzymatic andnon-enzymatic antioxidants. Ascorbic acid (Vitamin C) and α-tocopherol(Vitamin E) are two vitamins that belong to non-enzymatic class ofantioxidants.

When the reagent solution includes one or more antioxidants, suchantioxidants may act as singlet oxygen quenchers, free radicalscavengers, reducing agents or hydrogen donors. Suitable examples ofantioxidants may include ascorbic and iso-ascorbic acids and theirderivatives (such as ascorbates and iso-ascorbates, ascorbic acidpalmitate, stearate, and the like), tocopherols and trienols (alpha-,beta-, gamma- and delta-tocopherols and trienols), their derivatives andsynthetic soluble tocopherol analogs similar to Trolox, flavanoids(myricetin, quercetin, rutin, kaempferol, and the like), antioxidantphenolic compounds other than flavanoids (BHA, BHT, TBHQ tert-butylphenol, gallic acid, caffeic acid, and similar), thiols andhydrosulfites (mercaptoethanol, L-cystein, dithiothreitol, dithionite,dithioerythritol and the like), carotenoids (xanthopylls and carotenes),melatonin, uric acid and derivatives, ubiquinones and quinones,lactates, steroids, NADH and NADH analogs, calcium antagonists, vitaminB6, aspirin, steroids pyrrolopyrimidines, ebselen, metallothioneins,metalloporphyrins, antioxidant mixtures, and enzymatic reactive oxygenspecies (ROS) scavengers such as superoxide dismutase (SOD), superoxidereductases, catalase, thioredoxin reductase, alkenal/one oxidoreductase,and peroxiredoxins.

Ascorbic Acid and its Derivatives

For the purposes of the present invention, the following compounds maybe used as the additive in the reagent solution as described by formula(1)

wherein the additive is an ascorbic acid or a derivative thereofobtained by at least one substitution at position 2, position 3,position 5, position 6 or any combinations thereof with at least onegroup of atoms selected from a group consisting of an anion group ofatoms, an aliphatic group of atoms not exceeding 16 carbon atoms, and anaromatic group of atoms not exceeding 16 carbon atoms.

Ascorbic acid and its derivative the chemical structure of which areshown above exhibit powerful antioxidant activity in aqueous medium.Note that ascorbic acid is a compound of formula (1) in which R1, R2,R3, R4 are hydrogens. A number of ascorbic acid derivatives withsubstitutions at any one or a combination of positions 2, 3, 5 and 6retaining their antioxidant activity have been reported in theliterature. For the purposes of the invention, it is suggested to usethose ascorbic acid derivatives, which (i) retain their antioxidantproperties and (ii) present in aqueous solution in the forms describedbelow.

In embodiments, ascorbic acid derivatives may be those wherein one orseveral modifications of ascorbic acid are present: R1, R2, R3, and R4hydrogens may be substituted by groups of atoms selected from the groupconsisting of glucoside, glucopyranoside, phosphate, sulfate, lactate aswell as various alkanoyl ethers and esters. Non-limiting examples ofthese include compounds with carbon atom chains including anywherebetween 1 and 16 carbon atoms, such as for example ethyl, acetate,lactate, butyrate, caproate, octanoate, decanate, dodecanoate, oleate,myristate, palmitate, stearate and their mono- and poly-unsaturatedcounterparts.

These derivatives may encompass a variety of chemical entities rangingfrom simple salts (eg. Na, Mg, Ca, Fe, phosphate, sulfate) toderivatives such as O-substituted sugars (glucoside), as well as variousalkanoyl ethers and esters mentioned above.

Modification of ascorbic acid by substituting at the positions 2, 3, 5,or 6 of the ascorbic acid ring contributes to its stabilization as anantioxidant. Ascorbic acid derivatives may retain the same activityexhibited by ascorbic acid. For example, the antioxidant activity ispreserved for O-substituted ascorbic acid derivatives at position 2—suchas in ascorbic acid 2-glucoside, ascorbic acid 2-phosphate, and ascorbicacid 2-sulfate. The antioxidant activity is also preserved or evenincreased for various 6-O-alkanoyl-ascorbic acids such asascorbyl-6-octanoate, L-ascorbyl dodecanoate, ascorbyl palmitate,ascorbyl-2,6-dipalmitate, ascorbyl-5,6-dipalmitate andAscorbyl-6-stearate. The above mentioned compounds can be an L- or R- orracemic mixtures.

Further derivatives of ascorbic acid that may be useful for the purposesof the present invention include Alkali, alkaline-earth, transitionmetals (for example sodium ascorbate, (+)-Magnesium L-ascorbate, CalciumL-Ascorbate etc).

In addition, those derivatives of ascorbic acid are useful for use withthe assay of the invention which contain anion groups of atomsconsisting of halogens and other non-metals (for example2-Phospho-L-ascorbic acid trisodium salt, L-ascorbic acid 2-phosphate,Magnesium salt, L-ascorbic acid 2-sulfate, etc).

Aliphatic and/or aromatic groups or atoms as substitutes for positions2, 3, 5, 6 or combinations thereof also fall under the definition ofascorbic acid derivatives useful for the purposes of the invention. Insome embodiments, such groups of atoms may include anywhere between 1and 16 carbon atoms therein.

Examples of useful ascorbic acid derivatives containing aliphatic groupsof atoms with or without heteroatoms include 3-O-Ethyl ascorbic acid,Ascorbyl-2-glucoside, L-Ascorbic acid acetonide, Ascorbyl-6-octanoate,L-Ascorbyl Dodecanoate, Ascorbyl-6-palmitate, Ascorbyl-6-stearate,ascorbyl-2,6-dipalmitate and Ascorbyl-5,6-dipalmitate.

Examples of ascorbic acid derivatives containing aromatic group of atomsinclude 5-(phenylethynyl)uracil-2,3-di-O-benzylated 1-ascorbic acidderivative (as described by Gazivoda T et al. The novel C-5 aryl,alkenyl, and alkynyl substituted uracil derivatives of L-ascorbic acid:synthesis, cytostatic, and antiviral activity evaluations. Published inBioorg Med. Chem. 2007 Jan. 15; 15(2):749-58 incorporated herein byreference in its entirety).

Further examples of ascorbic acid derivatives with aromatic groups ofatoms include pyrimidine and purine derivatives of L-ascorbic acid (asdescribed by Raić-Malić et al. Novel pyrimidine and purine derivativesof L-ascorbic acid: synthesis and biological evaluation. J Med. Chem.1999 Jul. 15; 42(14):2673-8 incorporated herein by reference in itsentirety.

Finally, yet another group of derivatives of ascorbic acid may be usedfor the present invention, namely an O-alkyl ascorbic acid derivativescontaining at least one substitution of R3 at position 5, R4 at position6, or both R3 at position 5 and R4 at position 6 by a group of(CH₂)x-CH₃, where x is a number of (CH₂) elements, which may be 1 ormore but not exceeding 14.

It is also important to select the derivative of the ascorbic acid whichmaintains suitable solubility in an aqueous solution. Solubility isdefined herein as an ability of one compound to be present in thesolution and not precipitate. In order for the additive of the presentinvention to be effective, it may be present in the aqueous solution inat least one of the following forms: 1) the additive may be simplypresent in water, 2) the additive may be present in aqueous solutionsand dissolved in the form of amphiphilic structures such as micelles,liposomes and vesicles formed by another compound or 3) the additivesmay be present in aqueous solution in the form of self-assembledamphiphilic structures (supramolecular aggregates) such as micelles,liposomes and vesicles. Such supramolecular aggregates of ascorbic acidderivatives allow retention of the antioxidant activity of ascorbicacid. The examples of micelles formed by another than the additive ofthe present invention are micelles formed by detergents such asTween-20, Tween-80, Triton X-100, Cetyl trimethylammonium bromide(CTAB), and Sodium dodecyl sulfate (SDS). While ascorbic acid and itssalts are water-soluble, some ascorbic acid hydrophobic derivativescarrying hydrocarbon chains (e.g., ethers and esters at positions 2, 3,5, or 6 of the ascorbic acid ring) may form self-assembledsupramolecular aggregates such as micelles and vesicles and may bepresent in aqueous solutions in concentrations up to 50% (which is equalto about 1 to about 10 moles per liter). Since the effect of the presentinvention is generally observed at concentrations of additives inaqueous solutions starting from about 10×10⁻⁶ M and above, they may bepresent in an aqueous solution in one of the above described forms.

The term about is used herein and in other parts of the specification todescribe +/−25% variation of the cited value.

Other Compounds of the Reaction

Examples of suitable alcohols may include ethanol, methanol, glyceroland ethyleneglycol (HO—CH₂—CH₂—OH) and their polymer analogs such aspolyethyleneglycol.

Examples of suitable sugars may include monosacharides (erythrose,arabinose, ribose, glucose, mannose, xylose, fructose, galactose, andthe like), disaccharides (sucrose, maltose, cellobiose, lactose, and thelike), oligosaccharides (raffinose, stachyose, cyclodextrins, and thelike), and their polymer analogs polysaccharides (amylose, amylopectins,glycogen, cellulose, their derivatives, and the like).

Examples of suitable reactive oxygen species (ROS) may include ¹O₂, .O₂⁻, H₂O₂, ROO., OCl⁻, ONOO⁻, NO, and their unstable intermediates. Thefollowing reagents, which are known to be very effective scavengers ofhydroxyl radicals, may be selected for scavenging of hydroxyl radicals:ethanol, n-butanol, iso-propanol, 2-propanol and ethyleneglycol, DMSO,methional, sodium benzoate, sodium formate, mannitol.

Examples of suitable scavengers of superoxide anion O₂.⁻ may includesuperoxide dismutase (SOD), an enzyme that catalyzes dismutation ofsuperoxide to hydrogen peroxide.

Prior to irradiating a sample with light, one or a combination of theaforementioned reagents may be added.

The step of irradiating the enzymatic reaction components and additivesadded to these components with light may include providing such lightwithin a predefined range of wavelengths. In case of above describedDAP, the redefined range of wavelengths includes a visible lightspectrum. The time of exposure of the sample to irradiation with lightmay vary depending on light intensity.

One or more fluid samples may be irradiated simultaneously by a suitablesingle or plural source of light, such as an array of light emittingdiodes, a xenon lamp, a laser, a low power blue luminescent lamp, anarray of such lamps placed at a predefined distance between each other.In some embodiments, an air- or liquid-based cooling system such as acooling fan may be utilized. Activation of such cooling systems may bedone manually or automatically—for example based on the reading of atemperature sensor. Other accessories may also be utilized as known tothose skilled in the art.

One suitable detection technique is colorimetric detection using aphotometer or optical density reader. Using the PerkinElmer HIV-1 p24kit as an example, the absorbance or optical density (OD) of the samplemay be measured at 450 nm, which indicates the concentration of DAP, andtherefore the presence of HIV p24 antigen in the blood sample. Ofcourse, one skilled in the art would recognize that the OD may bemeasured at a wavelength that detects the presence of DAP formed in anyparticular assay method.

One or more acids may be added to the mixture after light irradiation asstop solutions. Such acids include, but are not limited to sulfuric acidor hydrochloric acid. The measuring wavelength of the DAP after additionof the stop solutions should be 492 nm.

Concentration of the final product of the photochemical amplificationreaction, such as DAP in the described example, may also be determinedby measuring fluorescence without or with addition of the fluorescenceconverter solution containing nonionic or ionic surfactants. Suitableexamples of surfactants may include polyoxyethylenesorbitanmonolaurate(Tween-20), octylphenoxypolyethoxyethanol (Triton X-100, andpolyethoxyethylene sorbitanmonooleat (Tween-80).

The enzymatic reaction of the assay of the present invention may proceedunder multiple conditions such as over various periods of time, ataltered temperature, pH, OPD concentration, or with the addition ofother additives disclosed herein. In an exemplary embodiment, theenzymatic reaction may proceed at room temperature in 0.1 Mphosphate-citric acid buffer, at a pH of about 5.0 with a totalenzymatic reaction time of about 30 minutes.

The increased production of DAP in the described example may be achievedwithout a corresponding increase in background noise. Therefore, theutility of the method is greatly increased over the prior art.

In accordance with another aspect of the invention, there is provided areagent kit or package of materials for accomplishing an assay of theinvention. The kit may include a solid support having an anti-analyteantibody attached to this support. The antibody may be selected to haveaffinity to the analyte. The kit may also include reference standardsfor the analyte, for example, one or more analyte samples of knownconcentration. The kit may further include other reagents or solutions,such as buffers, or labeled specific antigens, antibodies or complexesthereof useful in carrying out the assay. The kit may also contain areagent solution. The components of the kit may be supplied in separatecontainers, for example, vials, or two or more of the components may becombined in a single container.

In another aspect of the invention, described is an assay fordetermining the presence of an analyte. The analyte may be a HRPconjugate, a Hepatitis B Surface Antigen, an HIV p24 Antigen, a p50recombinant protein, a NKjBp50 homodimer, an RNA, a DNA, a mRNA, a cDNA,a Prostatic Specific Antigen, a Clostridium difficile toxin A, aClostridium difficile toxin B, a Rotavirus, an Anthrax (Bacillusanthracis), an Arenaviruse, a Clostridium botulinum toxin, a Brucellaspecie, a Burkholderia pseudomallei, a Chlamydia psittaci, a Vibriocholerae, an Ebola virus, an Escherichia coli O157:H7, a variola major,a Staphylococcal enterotoxin B, a Francisella tularensis, a Salmonella,a Rickettsia prowazekii, a Yersinia pestis, a Cryptosporidium parvum,and a Shigella. One skilled in the art would recognize that there aremany other substances including biological warfare, environmental- andfood chain-threat agents that can be determined using the method of thisinvention and thus the list above is not meant to limit the scope ofthis invention.

In another aspect of the invention, the PMI step may be used not onlyfor ELISA-type assays but also for other photosensitizer- orenzyme-mediated assays such as ligand-receptor assays, hybridizationassays, immunohistochemistry, in situ hybridization and membrane-basedassays: blotting analysis and immunochromatography tests. In theseenzyme-mediated assays, after conducting the conventional assay, the HRPconjugate may be detected using OPD substrate solution followed by thephotochemical amplification step of the invention as described above.

In another aspect of the invention, after conducting a conventionalassay, the HRP conjugate may be detected using OPD substrate solutionfollowed by the photochemical amplification step of the inventionincluding the use of other than OPD substrate and the reagent solutionas described above. In another aspect of the invention, OPD derivativesmay be used as a substrate of enzymatic catalytic reaction.

In another aspect of the invention, after conducting a conventionalassay, the HRP conjugate may be detected using other than OPD substratesolution during the enzymatic reaction (the product of which is aphotosensitizer), followed by the photochemical amplification stepincluding the use of either OPD substrate solution or other than OPDsubstrate and the reagent solution as described above.

In embodiments, the substrate of HRP-mediated reaction may be adye-tyramine complex, and the product of this reaction may be aphotosensitizer. Suitable photosensitizers may include afluorescein-tyramine, an eosin-tyramine and an erythrosine-tyramine.

Still additional steps of the invention may alternately includeconducting the enzymatic reaction on the surface of membranes, such asfor example nitrocellulose, nylon, and paper. In some aspects of theinvention, color changes or color spots caused by the addition of dyesor chemicals which result in the production of colors, may be viewed ordetected by photographing or by eye.

After conducting the enzymatic reaction on the surface of the membrane,such membrane may be washed with a rinsing buffer, and then thesubstrate solution containing the additives of the present invention maybe added. The reagent solution may comprise a mixture of ascorbic acidand a nonionic surfactant, or an alcohol and a nonionic surfactant, orany other combinations of the additives of the present invention asdescribed above. In still another step, the product of the enzymaticreaction may be taken out of the solution, placed in the substratesolution and irradiated by light.

The instant invention may have multiple uses. For example, one or moreof peroxidase molecules may be used as a label in enzyme immunoassay aswell as in blotting analysis or in immunohistochemistry. The peroxidaseactivity may also be detected by the method described in this inventionin assays including but not limited to phagocytosis, cell burst,oxidative capacity of cells and other methods known to those in the art.

Example 1 Determination of Diaminophenazine in Buffer Solution

In this system, various amounts of the photosensitizer Diaminophenazine(DAP) were added to the OPD substrate solution, and the solution wasilluminated. One can see that this system mimics a photosensitizer(dye)-mediated analytical assay, in which the amount of the analyte isdetermined. In this case, DAP, DAP derivatives or other photosensitizersmay be used as a label for the entities having an affinity to theanalyte to be detected.

The procedure for conducting photoamplification experiments withcompounds added to the OPD substrate solution was as follows. Briefly,30 μl of the reagent solution (additives) was added to the 100 μl of OPDsubstrate solution (0.1 M phosphate-citrate buffer, pH 5.0), containingvarious amounts of DAP as a primer. After 30 min incubation, the sampleswere irradiated by visible light with wavelengths between 400 nm and 500nm for various times using a “high” power Device 1 for even illuminationof large surfaces, high power defined as between 1.0 and 2.0lumen/centimeter²/steradian light intensity. Then, the optical densityof the samples was measured using a standard spectrophotometer orstandard optical density reader.

FIG. 1A shows calibration (standard) curves for the detection of DAPprimer added to the OPD substrate solution in double dilution at varioustimes of the illumination in the absence of additives. As can be seen inFIG. 1A, the background (no DAP added) increases with the illuminationtime significantly. This feature of the DAP calibration curves affectsthe performance of the assay because in this case limit of detection(LOD) of the assay, defined as double the value of the backgroundsignal, increases with the illumination time.

In order to circumvent this undesirable effect, some additives which arethe inhibitors or retarders of the photochemical photosensitizedreactions may be used (see above). FIG. 1B illustrates the effect ofascorbic acid (AA) on the kinetics of the DAP-mediated photosensitizedreaction. In the presence of AA the increase of the background withillumination time is significantly lower than that when no additives areadded to the OPD substrate solution. A similar effect was observed usingderivatives of AA, glycerol, ethyleneglycol and polyethyleneglycols (seeExample 3).

FIG. 1C shows the calibration curves for determination of DAP when areagent solution containing 250 μM of AA is added. As can be seen inthis Figure, the background increase is limited, and the detection limit(analytical sensitivity) of the assay is significantly lower than thatfor the assay with no additives added (see FIG. 1A). The same effect canalso be achieved using a reagent solution containing 500 μM AA and 1.5%Tween-20 (FIG. 1D). The addition of the surfactant Tween-20 may lead toincrease of the signal and decreasing of the illumination time. As canbe seen in FIGS. 1C and 1D, the addition of the reagent solutionscontaining temporary inhibitor (retarder) of the photochemical reaction(ascorbic acid) leads to drastic decrease of the limit of detection ofthe determination of the photo sensitizer, DAP.

Indeed, the detection limit defined as double the value of thebackground of DAP determination depends on the background value and onthe slope of the calibration curve. That is, the lower the backgroundand higher the slope, the lower the detection limit of the analytedetermination. The detection limit of the conventional assay withoutphotochemical amplification can be estimated as 1:1 DAP dilution (FIG.1A, 0 min illumination curve), whereas the detection limit of the assaywith photochemical amplification when no reagent solution added (PAM,(method without the use of the reagent solution with additives of thepresent invention, FIG. 1A) is approximately 1:8 DAP dilution (FIG. 1A,4 min illumination curve). The addition of the reagent solutionscontaining AA (or some other inhibitors and retarders of thephotochemical reaction) to the OPD substrate solution changes thecharacter of calibration curves for DAP determination drastically (FIGS.1C and 1D), and as a consequence leads to decrease of the detectionlimit of the assay. Indeed, a more than 32- and 16-fold decrease of thelimit of detection of DAP using a photochemical amplification with thereagent solution containing the additives of the present invention (PMI)(FIGS. 1C, 16 min illumination curve and 1D, 12 min illumination curve,respectively) occurs as compared to the PAM (method without the use ofthe reagent solution with additives of the present invention, FIG. 1A)and 256- and 128-fold increase as compared to the conventional assay(FIG. 1A).

Example 2 Determination of HRP Concentration in Buffer Solution

In this system, HRP conjugate concentration in the buffer solution isdetermined using the prepared calibration curves. In order to preparethe calibration curves for determination of HRP, OPD substrate solutionwas added to the HRP conjugate solutions at various concentrations, andafter incubation, the solutions were illuminated for a certain period oftime. One can see that this system mimics the last step ofenzyme-mediated analytical assays, in which the amount of HRP bound tothe well of the microtiter plate (as in ELISA-type assays) or membrane(as in blotting analysis) or cell surface (as in immunohistochemistry orin situ hybridization) is determined.

In more detail, to each test well of a standard 96-well microtiter platecontaining 20 of the HRP conjugated antibody (PerkinElmer) at variousdilutions in 0.01 M phosphate buffer, pH 7.0, 80 μl of OPD substratesolution was added. The HRP-mediated oxidation of OPD was carried out atroom temperature in 0.1 M phosphate-citric acid buffer at pH 5.0. Thereaction time was 30 min in all experiments. 30 μl of reagent solutions(additives) were added to the OPD substrate solution after theincubation step before illumination. After incubation period thesubstrate solution was irradiated using the illumination instrument forirradiation of large surfaces (Device 1, high power). The opticaldensity of the samples at 450 nm was measured using an optical densitymicrotiter plate reader.

In FIG. 2A, the optical density of the background (no HRP in the sample)for the conventional procedure is in the range of 0.03-0.05. Themeasurement of the optical density by regular commercially availablereaders has an error on the order of ±0.01. In such cases, companiesmanufacturing diagnostic analytical kits define the detection limit ofthe assay relying on practical assumptions. In particular, thereasonable threshold value of the signal of the assay should be defined.In our case, the reasonable threshold signal can be equal toBackground+0.03 for the conventional enzymatic determination. Therefore,as can be seen in FIG. 2A, 0 min illumination curve, the detection limitof the conventional assay is 1:40,000 dilution of the HRP conjugate.FIG. 2A also shows that the detection limit for HRP determination by theassay with photochemical amplification with no reagent solutioncontaining additives of the present invention (PAM) at 2 and 4 minutesillumination is approximately 1:320,000 HRP conjugate dilution which is8-fold lower than that of the conventional assay procedure (withoutillumination). One of the features of the FIG. 2A is that the slope ofthe HRP calibration (standard) curves decreases with increasing of theillumination time. This affects the performance of the assaysignificantly. As we mentioned above, the detection limit defined astwice the value of the background of HRP determination depends on thebackground value and on the slope of the calibration curve. That is, thelower the background and higher the slope, the lower the value ofdetection limit of the analyte determination.

The addition of the reagent solution containing 250 μM ascorbic acid inphosphate-citrate buffer (PCB), pH 5.0 (reagent solution #1) to the OPDsubstrate solution after conducting the enzymatic step changes thecharacter of the calibration curves for HRP determination (FIGS. 2B-D),and results in a drastic decrease of the detection limit of the assay.As can be seen in FIG. 2B, 14 and 16 min illumination curves, thedetection limit of the assay can be estimated to be1:5,120,000-1:2,560,000 of the HRP dilution, which is 128-64-fold lessthan the limit of detection of HRP conjugate as compared to theconventional procedure, and approximately 16-8-fold less than thedetection limit of the PAM. FIG. 2C shows the effect of adding thereagent solution containing 250 μM ascorbic acid and 1.5% Tween-20 inPCB (reagent solution #2), which results in decrease in the limit ofdetection of the assay and increase in the rate of the photochemicalreaction. One can further limit the increase of the background signal byusing the reagent solution #3 containing as twice the ascorbic acidconcentration as compared to the reagent solution #2 (FIG. 2D).

Example 3 Determination of HRP Concentration Using Reagent SolutionsContaining Ascorbic Acid Derivatives and Materials PossessingAntioxidant Activity

Calibration curves for determination of HRP were prepared in the samemanner as described in the Example 2, except that reagent solutions thatwere added after performing the incubation step before illuminationcontained other than ascorbic acid compounds. The obtained results arepresented in FIGS. 3A-C.

As can be seen in FIG. 3A, 10 min illumination curve, the addition ofthe reagent solution containing 125 μM ascorbic acid 6-palmitate and1.5% Tween-20 in PCB to the OPD substrate solution results in decreaseof the limit of detection (LOD) of the assay, which is between1:5,120,000 and 1:2,560,000 of the HRP conjugate dilution, as comparedto the conventional assay 64-128 fold. Comparable increase in theanalytical sensitivity (decrease in LOD) was observed when the reagentsolutions containing 250 μM isoascorbic acid+1.5% Tween-20 in PCB (FIG.3B, 12 min illumination curve) and 18.6% glycerol+1.5% Tween-20 in PCB(reagent solution #4, FIG. 3C, 14 min illumination curve) were used.

Similar results were obtained using ethyleneglycol andpolyethyleneglycols. We assume that some materials such as glycerol,ethyleneglycol and polyethyleneglycols may have reducing and antioxidantactivity or may contain antioxidants as impurities. It should beemphasized that the addition of the aforementioned reagent solutionsbefore performing enzymatic reaction leads to the decrease of the assayLOD as well.

Example 4 Preparation of the Calibration Curve for Determination ofClostridium difficile Toxin A and Toxin B by PMI

Clostridium difficile bacteria is related to the same class ofpathogenic bacteria as Clostridium botulinum, biological warfare agent.Cytoclone A+B ELISA kit reagents for determination of Clostridiumdifficile toxin A and toxin B were obtained from the Cambridge Biotech.Conventional ELISA procedure for the determination of the above antigenswas performed according to the manufacturer's procedure. Briefly, thecalibration curves for the Clostridium difficile toxin A and toxin Bwere prepared as follows: to each test well 100 μl of appropriate HRPconjugated antibody and 100 μl of negative and positive controls invarious dilution were added. The mixtures were incubated for 60 minutesat 37° C. for toxin A+B detection. Wells were rinsed 5 times withwashing buffer, and 100 μl of TMB substrate solution was added to eachwell. After 20 minutes incubation, the reaction was terminated with 50μl of stopping solution. The absorbance at 450 nm was read using theTiterscan Multiscan reader.

Preparation of the calibration curves for the determination of the aboveantigens with PMI step involved carrying out the slightly modifiedconventional ELISA procedure and irradiation of the samples as describedabove. The modification of ELISA procedure was just in that OPDsubstrate solution was used instead of TMB substrate solution. Afterenzymatic reaction was finished, the reagent solution containing 500 μMAA and 1.5% Tween-20 in phosphate-citrate buffer was added to the OPDsubstrate solution. Then, samples being in 96-well microtiter plate wereirradiated for various time and optical density or fluorescence of thesamples was measured as described above.

The standard curves prepared by conventional ELISA and ELISA with PMIfor Clostridium difficile toxin A and toxin B determination in the rangeof positive control dilutions from 1:320 to 1:10 are shown in the FIG.4. According to the protocol of Cambridge Biotech Co. the cut-off linefor detection of these antigens by the conventional ELISA procedure is0.2 optical density values. Therefore, the detection limit for thismethod equals approximately to 1:5 positive control dilution. As can beseen in FIG. 4, the detection limit for toxin A+B determination usingPMI equals to 1:300 dilution of the positive control. Thus, thesensitivity of the assay increases more than 50-fold when PMI is used.

Example 5 Preparation of the Calibration Curve for Determination of HIVp24 Antigen by PMI

An ELISA kit for detection and quantification of HIV p24 antigen wasobtained from Perkin Elmer Life Sciences (Boston, Mass.). This kitcontains a 96-well microtiter plate the wells of which are coated with ahighly specific mouse monoclonal antibody to HIV-1 p24. The immobilizedmonoclonal antibody captures both free HIV-1 p24 and that which has beenreleased upon disruption of immune complexes in the serum/plasma sample.The captured antigen is complexed with biotinylated polyclonal antibodyto HIV-1 p24, followed by a streptavidin-HRP (horseradish peroxidase)conjugate. The resulting complex is detected by incubation withortho-phenylenediamine-HCl (OPD), which produces a yellow color that isdirectly proportional to the amount of HIV-1 p24 captured. Theabsorbance of each microplate well is determined using a microplatereader and calibrated against the absorbance of an HIV-1 p24 antigenstandard or standard curve. Samples with absorbance values equal to orgreater than the cutoff factor are considered initially reactive.

Conventional Non-ICD ELISA.

The HIV p24 ELISA test was performed in accordance with themanufacturer's instructions. Analytical sensitivity of the conventionalELISA test was determined via the least squares fit to the standardcurve at an absorbance equal to the cutoff defined by the manufacturer(i.e., mean negative control OD+0.050). The conventional ELISAcalibration curves for quantification of HIV-1 p24 antigen were providedby the manufacturer and prepared by us. The detection limit wascalculated by us as recommended by the manufacturer and by usingregression analysis, and showed that they are equal to 3.5 and 4.5pg/mL, respectively. These values are in agreement with that provided bythe manufacturer for the detection limit of the conventional non-ICDELISA test for HIV-1 p24 antigen, which is 3.5 pg/mL. The detectionlimits were also estimated as the analyte concentration corresponding tothe twice the value of the background signal, and practically the sameresults were obtained. The calibration curve prepared for theconventional non-ICD ELISA test is presented in FIG. 5A.

Non-ICD p24 ELISA+PMI. In order to reach the maximum sensitivity of theELISA+PMI, the conditions for performing the conventional assay werefirst optimized. This is because the conventional test conducted withoutany changes is rather “noisy”, that is, it is characterized by a highlevel of non-specific binding of reagents. In the non-ICD ELISA+PMIassay, a high background signal may affect the performance of the testbecause the amplification of both signal and background may occur.Therefore, in order to reach the maximum signal-to-noise ratio inELISA+PMI, the background (noise) signal should be as low as possible.With respect to this, the procedure for carrying out the conventionalELISA-based test was slightly modified. The assay steps were the same asfor the conventional assay procedure described above, except decreasedincubation time (10 min instead of 30 min in conventional assay) withOPD substrate solution was used. After a 10 min incubation step, 30 μlof the reagent solution #1 (250 μM ascorbic acid in PCB), was added to100 μl of OPD substrate solution, and samples were irradiated using apowerful illumination Device 1. Calibration curves for determination ofp24 antigen were prepared by double dilution of positive control innegative control (normal human serum). The calibration curves preparedusing non-ICD ELISA+PMI under the above conditions and at various timesof illumination are presented in FIG. 5B. The detection limit calculatedas the analyte concentration corresponding to twice the value of thebackground signal using a regression analysis for the p24 antigenELISA+PMI at 12 min of illumination, is equal to 0.08 pg/mL. Thus, theanalytical sensitivity of the ELISA+PMI is approximately 44-fold higherthan that for the conventional assay.

Heat-mediated ICD ELISA (Ultrasensitive ELISA without tyramide signalamplification) was suggested by us to increase the sensitivity of ICDELISA+PMI as we did not receive significant signal amplification whenusing PMI with conventional Perkin Elmer ICD ELISA. The complexdisruption and ELISA protocols from Ultrasensitive HIV-1 p24 assay(PerkinElmer) were adopted, but the tyramide signal amplification stepwas excluded. The calibration curve prepared for heat-mediated ICD ELISAtest is presented in FIG. 5C. The detection limit for the heat-mediatedICD ELISA was calculated by regression analysis, and is equal to 40pg/mL.

Heat-mediated ICD p24 ELISA+PMI. Heat-mediated ICD p24 ELISA steps werethe same as for Ultrasensitive HIV-1 p24 assay (PerkinElmer), exceptthat four changes were made: 1) incubation with the detector antibodywas carried out for 2 hours instead of 1 hour; 2) incubation time withthe decreased amount of Strep-HRP conjugate (1:400 dilution) was 30 minat room temperature; 3) tyramide signal amplification step andincubation with Strep-HRP (FP105) were omitted. After a 30 minincubation enzymatic step of OPD oxidation, 30 μl of the reagentsolution #1 (250 μM ascorbic acid in PCB), was added, and samples wereirradiated as described above. The calibration curves prepared usingheat-mediated ICD ELISA+PMI under the above conditions and at varioustimes of illumination are presented in FIG. 5D. The detection limitcalculated as the analyte concentration corresponding to twice the valueof the background signal for the p24 antigen ICD ELISA+PMI at 8 min ofillumination, is equal to 0.15 pg/mL. Thus, the analytical sensitivityof the ELISA+PAM is more than 100-fold higher than that for the assaywithout signal amplification. It is worth noting that the sensitivity ofthe heat-mediated ICD ELISA+PMI is approximately 12 times higher thanthat for PerkinElmer Ultrasensitive p24 assay with tyramide signalamplification.

Example 6 Preparation of the Calibration Curve for Determination of NFkBp50 Homodimer by PMI

An ELISA-based kit for detection and quantification of transcriptionfactor activation was obtained from ActiveMotif (Carlsbad, Calif.). Inour studies, we used a kit for determination of NFκB p50 homodimer. Thiskit contains a 96-well plate to which oligonucleotide containing an NFκBconsensus-binding site has been immobilized. The Jurkat (TPA+CI) nuclearextract and p50 recombinant protein are provided as positive controlsfor NFkB p50 activation. The activated NFkB contained in nuclear extractor p50 recombinant protein specifically bind to theoligonucleotide-coated plates. By using an antibody that is directedagainst either the NFkB p50 subunit, the NFκB complex bound to theoligonucleotide is detected. Addition of a secondary antibody conjugatedto horseradish peroxidase (HRP) provides sensitive colorimetric readoutthat is easily quantified by spectrophotometry. The 96-well plate withindividual strips of 8 wells is suitable for manual use orhigh-throughput screening applications.

The p50 NFkB ELISA-based test was performed in accordance with themanufacturer's instructions. For the p50 NFkB determination by theELISA+PMI, the assay steps were the same as for the conventional assayprocedures described above, except 1) Decreased amount of HRP-conjugatedantibodies were used (see below), and 2) 100 μl of the OPD substratesolution was added to each test well instead of TMB substrate solution.After incubation, 30 μl of the reagent solution #2, containing 250 μMascorbic acid and 1.5% Tween-20, was added, and samples were irradiatedusing a moderate power Device 2 for even illumination of large surfaces,moderate power defined as between 0.3 and 1 lumen/centimeter²/steradianlight intensity. Calibration curves for determination of p50 recombinantprotein and activated cell extracts were prepared by double dilution ofcorresponding positive controls in buffers recommended by themanufacturer. The detection limit of the tests was estimated as theanalyte concentration corresponding to twice the value of the backgroundsignal. In all experiments, the intra-assay variations did not exceed10%. This indicates that the obtained data are reliable andreproducible.

The calibration curve for quantification of p50 recombinant proteinprepared using the conventional ELISA-based method is shown in FIG. 6A.The detection limit for the conventional assay is equal to approximately0.4 ng/well. This value is in agreement with that provided by themanufacturer for the detection limit of the conventional ELISA-basedtest for p50 recombinant protein. The calibration curves prepared usingELISA+PMI at various times of illumination are presented in FIG. 6B. Thedetection limit calculated using a regression analysis for the p50recombinant protein ELISA+PMI at 6 min of illumination, is equal to0.005 ng/well. Thus, the analytical sensitivity of the ELISA+PMI isapproximately 80-fold higher than that for the conventional assay.

The calibration curves for determination of NFκB transcription factor incell extracts with the NFκB p50 kit using the conventional ELISA-basedtest and ELISA+PMI are shown in FIGS. 6C and 6D, respectively. Thedetection limits calculated as described above are 0.5 μg/well and 0.03μg/well for the conventional and ELISA+PMI tests, respectively. Thus,the sensitivity of the assay increases approximately 15-fold, ascompared to that of the conventional ELISA-based test.

Example 7 The Use of the Modified PAM for Increasing of the Sensitivityof Commercially Available AMPLICOR HIV MONITOR Test, Version.1.5

Materials and Methods.

Diagnostic kit for determination of HIV load was obtained from RocheDiagnostics Corporation (Indianapolis, Ind.).

Conventional Method:

The AMPLICOR HIV-1 Monitor test, an RT-PCR with an internal quantitationstandard, was performed in accordance with the manufacturer'sinstructions (Roche, 2003). Briefly, this test is based on five majorprocesses: specimen preparation; reverse transcription of target RNA togenerate complementary DNA (cDNA); PCR amplification of target cDNAusing HIV-1 specific complementary primers: hybridization of theamplified products to oligonucleotide probes specific to the target(s);and detection of the probe bound to amplified products by colorimetricdetermination using HRP-avidin conjugate.

The AMPLICOR HIV-1 Monitor test permits simultaneous reversetranscription and PCR amplification of HIV-1 and HIV-1 QuantitationStandard RNA. The Master Mix reagent contains a primer pair specific forboth HIV-1 and HIV-1 Quantitation Standard RNA and has been developed toyield comparable quantitation of group M subtypes of HIV-1.

The quantitation of HIV-1 viral RNA was performed using the HIV-1Quantitation Standard. The HIV-1 Quantitation Standard is anon-infectious RNA transcript that contains the identical primer bindingsites as the HIV RNA target and a unique probe binding region thatallows quantitation standard amplicon to be distinguished from HIV-1amplicon. The Quantitation Standard is incorporated into each individualspecimen at a known copy number and is carried through all theaforementioned procedures along with HIV-1 target and is amplifiedtogether with the HIV-1 target. HIV-1 RNA levels in the test specimensare determined by comparing the HIV-1 signal to the QuantitationStandard signal for each specimen (see below). The Quantitation Standardcompensates for effects of inhibition and controls for the amplificationprocess to allow the accurate quantitation of HIV-1 RNA in eachspecimen. The stated limit of detection is 400 copies/ml and lineardynamic range is 400 to 750,000 copies/ml.

PMI Procedure:

PCR+PMI consists of two steps: conventional procedure and PMI. InPCR+PMI, the conventional method was performed without changes exceptthe originally used TMB (tertramethylbenzidine) HRP substrate solutionwas replaced by the OPD substrate solution. Once the enzymatic reactionwas finished, the 30 μL of the reagent solution #4 (18.6% glycerol and1.5% Tween-20 in PCB) was added to 100 μL of the OPD substrate solution.Samples in 96-well microtiter plates were irradiated for various timesusing “low” power Device 3 for even illumination of large surfaces, lowpower defined as between 0.1 and 0.3 lumen/centimeter²/steradian lightintensity. Optical density of the samples was measured using a BioRadmicrotiter plate reader (California) at 450 nm or 492 nm after additionof stopping reagent (2M H₂SO₄).

In order to show the feasibility of using PMI for increasing of theanalytical sensitivity of HIV test, a low positive control (HIV-1 L(+)control) provided with the AMPLICOR HIV-1 Monitor test kit was used. Lowpositive control contains non-infectious in vitro transcribed RNA(microbial) containing HIV-1 sequences in concentration from 980 to8,900 copies/ml. In order to study the effects of dilution of lowpositive control before performing of PCR reaction (which models thereal-life experiment with clinical samples) and the kinetics of thephotochemical amplification of signals, double dilutions of low positivecontrol at starting dilutions 1:16 and 1:64 were prepared. Since thecurves prepared for low positive control at these two starting dilutionspractically coincide with each other, in FIG. 7A only the ODs obtainedfor low positive control at starting dilution 1:64 are presented. FIG.7B shows the results obtained for Quantitation Standard at differenttimes of illumination.

As can be seen in FIG. 7A, the detection limit of PCR+PMI isapproximately 1:512 dilution of the low positive control. The detectionlimit of the conventional assay in this experiment was determined to bea 1:25 dilution of the low positive control. Results demonstrate thatthe limit of detection for HIV using PCR+PMI can be decreased at least20-fold as compared to the conventional method. As can be seen in FIG.7B, ODs of the quantitation standard is changing with illumination andhave to be taken into consideration when calculating HIV-1 viral load(see below).

In order to examine the possibility of using PCR+PMI for quantitativedetermination of HIV load in clinical samples, values of HIV load in lowpositive control and several clinical samples by the conventional methodand by PCR+PMI we calculated, and the obtained results were compared.

The AMPLICOR HIV-1 RNA MONITOR Test v.1.5 quantitates viral load byutilizing a second target sequence (HIV-1 Quantitation Standard (QS))that is added to the amplification specimen at a known concentration.The QS is a non-infectious 233 nucleotide in vitro transcribed RNAmolecule with primer binding region identical to those of the HIV-1target sequence. The QS, therefore, contains SK145 and SKCC1B primerbinding sites and generates a product of the same length (155 bases) andbase composition as the HIV-1 target. The probe-binding region of the QSwas modified to differentiate QS-specific amplicon from HIV-1 targetamplicon.

In the linear range of the assay, the optical density in each well ofthe plate is proportional to the amount of HIV-1 or QS amplicon in thewell. Total OD is calculated by multiplying the OD in each well by thedilution factor for that well. The calculated total HIV-1 OD or total QSOD is proportional to the amount of HIV-1 or QS RNA, respectively,present in each reverse transcription/PCR amplification reaction. Theamount HIV-1 RNA in each specimen is calculated from the ratio of thetotal optical density for the HIV-1 specific well to the total opticaldensity for the QS-specific well and the input number of QS RNAmolecules using the following equation:

${\lbrack \frac{{Total}\mspace{14mu}{HIV}\text{-}1\mspace{14mu}{OD}}{{Total}\mspace{14mu}{QS}\mspace{14mu}{OD}} \rbrack \times {Input}\mspace{14mu}{QS}\mspace{14mu}{copies}\mspace{14mu}{per}\mspace{14mu}{PCR}\mspace{14mu}{reaction} \times \mspace{281mu}{Sample}\mspace{14mu}{volume}\mspace{14mu}{factor}} = {{HIV}\text{-}1\mspace{14mu}{RNA}\mspace{14mu}{copies}\text{/}{ml}}$where Total HIV-1 OD is the calculated Total OD for HIV-1 amplicon,Total QS OD is the calculated Total OD for HIV-1 QS amplicon, Input QScopies per PCR reaction is the number of copies of QS in each reaction;this information is lot-specific, Sample volume factor is the factor toconvert copies/PCR to copies/ml, and usually equals 40.

In more detail, the specimens in neat and 1:5, 1:25, 1:125, 1:625 and1:3125 serial dilutions are added to the wells from A to F containing acomplementary oligonucleotide to HIV-1 amplicon. The specimens in neatand 1:5 dilutions are added to wells G and H containing a complementaryoligonucleotide to QS amplicon. Every plate contains a negative control(negative human plasma). Then, the HIV-1 OD above and closest to 0.2,and QS OD above and closest to 0.3 are selected for the furthercalculation: The total HIV-1 OD and total QS OD are calculated bymultiplying the background-corrected OD value (OD (sample or QS)−OD(negative control)) by the dilution factor associated with that well,and the above formula is used for the calculation of HIV-1 load.

The incorporation of QS to each specimen is a necessary step in order tomitigate the variations in the performance of several test procedures.It should be noted that the above original approach for the calculationof HIV load has certain drawbacks. Indeed, the requirement for selectingof HIV and QS ODs above and closest to 0.2 and 0.3, respectively, iscaused by the fact that the kinetics of enzyme-catalyzed increase of thesignals is not linear, and as a consequence the final ODs are notproportional to the initial concentration of the analytes (amplicons).Since only two concentrations of QS are used in the assay, the selectedODs could differ significantly from each other, and due to non-linearityof the signal increase, the errors in the calculation of HIV load couldbe considerable.

The HIV and QS OD values closest to each other and above OD=0.2 wereselected for the further HIV load calculation. In this case, variationscaused by the non-linear signal increase of enzymatic reaction andphotoamplification will be minimized. Initially, in PCR+PMI the aboveoriginal protocol for calculations of HIV load was used. However, moreaccurate values of HIV-1 load can be obtained using a slightly modifiedprotocol. The following example illustrates the suggested approach.

Three clinical samples containing HIV virus in different concentrationswere used. Conventional and PCR+PMI were performed, and HIV-1 load usingthe above original and modified protocols have been calculated. In orderto compare the results obtained using the conventional method andPCR+PMI, samples containing HIV virus in concentrations that could bedetected by the conventional procedure were used. In PCR+PMI, however,we diluted samples in negative human plasma until the level when HIVload was undetectable by the conventional test, that is samples PS, TJSand CAP-5-05 were used at starting dilutions 1:200, 1:400 and 1:100,respectively. PCR+PMI was performed without any changes in theconventional procedure, except in PCR+PMI, dilutions of quantitationstandard were 1:10 and 1:50 whereas in the conventional method they were1:1 and 1:5.

In FIG. 7C, the results obtained for clinical sample PS at startingdilution 1:200 are presented. As can be seen in this Figure, the ODsobtained for PS and QS are increasing with increasing the illuminationtime. The similar graphs were obtained for other clinical samples. Usingthe data from these graphs, HIV load in these specimens at differenttimes of illumination using a modified protocol was calculated. Theresults are summarized in FIG. 7D. For each sample the values of HIVload calculated at different times of illumination are practically thesame, and the coefficient of variation does not exceed 10%. Theseresults show that HIV load determination does not depend on illuminationtime. Note that in the photoamplification method, it is impossible touse the original protocol for the calculation of HIV load due to thefact that the calculated values of HIV load depend significantly onillumination time. Therefore, in PCR+PMI only the modified protocol forthe calculation of HIV load was used. It should also be noted thatdifferent lots of Low positive samples having different viral loads wereused for obtaining the results presented in FIGS. 7A and 7D.

FIG. 7E summarizes the average values of HIV loads (and correspondingcoefficients of variation) for low positive control and clinical samplesobtained at different starting dilutions of samples and different days.Results demonstrate that there is a good agreement with the resultsobtained by the conventional method and PCR+PMI. The coefficients ofvariation are rather high for both conventional and PCR+PMI. This is notunusual because according to a much more detailed study carried out bythe manufacturer of this kit, coefficients of variation in the linearrange for this kit were between 30 and 94%. It should be emphasized thatfor the conventional method there is no significant difference betweenthe values of HIV load and coefficients of variation calculated usingthe original and modified protocols. Therefore, both protocols are validfor the calculation of HIV load in the samples.

Example 8 The Use of OPD as an Insoluble HRP Substrate in Membrane-BasedAssay (Blotting Analysis)

1. Comparison of Insoluble Substrates for HRP-Mediated Membrane Assays

Chloronaphtol (4-CN), 3-aminoethylcarbazol (AEC), HRP-conjugated goatantihuman antibodies and polyclonal human antibodies were purchased fromSigma. TMB soluble and insoluble substrate solutions were obtained fromTransgenic Sciences, Inc (Massachusettes) and Calbiochem-NovabiochemCorp. (La Jolla, Calif.), respectively. Tablets containing 12.8 mgorthophenylenediamine dihydrochloride were purchased from AbbottLaboratories (North Chicago, Ill.). Nitrocellulose membranes with 0.45μm pores and Nylon membranes “Hybond” were obtained from GE HealthcareLife Sciences (Piscataway, N.J.). To prepare the OPD substrate solution,a tablet containing 12.8 mg OPD (Abbott) was dissolved in 10 mL of 0.1Mphosphate-citrate buffer, pH 5.0 containing 0.01% hydrogen peroxide.4-CN and AEC substrate solutions were prepared according to standardpreviously published procedures (Jackson and Blythe, 1993). Commerciallyavailable insoluble TMB substrate solution (Calbiochem-Novabiochem) wasused as received.

To show the possibility of using OPD as an insoluble HRP substrate inmembrane-based (blotting) analysis and compare the efficacy of differentHRP insoluble substrates, serial double dilutions of HRP-conjugatedantibodies were made in 0.01M phosphate buffered saline (PBS), pH 7.2.Two μl aliquots were spotted on the nitrocellulose or nylon membranestrips. The membrane strips were air-dried and then immersed for 30minutes in freshly made up chromogenic substrate solutions in theirappropriate buffers. The strips were then removed from the chromogenicsubstrate solutions, washed five times with distilled water, air-driedand photographed. Representative results using 4-CN, AEC, TMB and OPD assubstrates for HRP on nitrocellulose membrane are shown in FIG. 8A. Ascan be seen in this Figure, the detection system using OPD as an HRPsubstrate is more sensitive than systems using 4-CN and AEC and offerssensitivity comparable to that of the system employing a commercial TMBinsoluble substrate solution. Practically the same results for wereobtained using “Hybond” nylon filters (see FIG. 8B, strip 2).

FIG. 8B demonstrates that blocking of nitrocellulose or nylon membranestrips with 5% BSA after spotting a various amounts of HRP-conjugatedantibodies does not prevent the OPD oxidation product to be adsorbedonto the strips surface. OPD was added to the strips 1 (nitrocellulose)and 2 (nylon) without blocking the membranes with BSA. Strips 3(nitrocellulose) and 4 (nylon) were blocked with 5% BSA after spottingvarious amounts of HRP-conjugated antibodies. As can be seen in FIG. 8B,the assay sensitivities with OPD for the strips unblocked and blockedwith BSA are between 16,000-32,000 dilution of the stock HRP-conjugatesolution.

2. Application of the PMI Method to Membrane- and Histochemistry-BasedAssays

In order to show the possibility of using the Photochemical Method ofthe present invention (PMI) for membrane-based assays, serial dilutionsof HRP-conjugated antibodies were prepared in 0.01M phosphate bufferedsaline (PBS), pH 7.2. Two μl aliquots were spotted on the nylon membranestrips. The membrane strips were air-dried and then immersed for 30minutes in freshly made up OPD chromogenic substrate solution describedabove. The strips were then removed from the chromogenic substratesolution, washed five times with distilled water. Then, the strips wereimmersed into OPD substrate solution containing 250 μM ascorbic acid and1.5% Tween 20 for 5 min. After this, they were taken off the solutionand without washing placed on the glass surface. Then, they wereilluminated for 5 min from above using the device for even illuminationof large surfaces. The results are shown in FIG. 8C. As can be seen inFIG. 8C, the sensitivity of the assay plus PMI is approximately1:800,000 dilution of the stock HRP solution, which is approximately30-fold higher than that for the conventional procedure with no PMI(˜1:25,000).

Examples 9-11

The procedure of Example 2 was repeated except that ascorbic acid orascorbic acid+1.5% Tween 20 was replaced by the following additives:

Example 9

1 mM or 0.5 mM L-Ascorbic acid acetonide or 1 mM or 0.5 mM L-Ascorbicacid acetonide+1.5% Tween 20;

Example 10

1 mM or 0.5 mM L-Ascorbyl octanoate or 1 mM or 0.5 mM L-Ascorbyloctanoate

+1.5% Tween 20;

Example 11

1 mM or 0.5 mM L-Ascorbyl dodecanoate or 1 mM or 0.5 mM L-Ascorbyldodecanoate+1.5% Tween 20.

The increase in the analytical sensitivity of the assays fordetermination of HRP (catalyzing the photosensitizer DAP) in aqueoussolution using various additives was calculated and shown to have beenincreased from about 50- to about 100-fold as compared with conventionalprocedure.

Example 12

The procedure of Example 1 was repeated except that the photosensitizerDAP was replaced by a photosensitizer Eosin Y (starting concentration is10 μM) and 100 μM, 50 μM, 20 μM and 10 μM sodium ascorbate was used asan additive. The improvement in signal/background ratio was measured andshown to be from about 3- to about 5-fold at 100 μM sodium ascorbate ascompared to when no additive was used.

Example 13

The procedure of Example 1 was repeated except that photosensitizer DAPwas replaced by photosensitizer Eosin Y (starting concentration is 10μM), substrate OPD was replaced with the substrate TMB and 50 μM, 20 μMand 10 μM sodium ascorbate was used as an additive. The improvement insignal/background ratio was measured and it is equal to from 2- to3-fold at 50 μM sodium ascorbate as compared to when no additive wasused.

REFERENCES CITED

-   Arababadi, M. K., Hassanshahi, G., Pourfathollah, A. A.,    Zarandi, E. R. and Kennedy, D. (2011) Post-Transfusion Occult    Hepatitis B (OBI): A Global Challenge for Blood Recipients and    Health Authorities. Hepat Mon 11, 714-8.-   Avrameas, S, and Uriel, J. (1966) [Method of antigen and antibody    labelling with enzymes and its immunodiffusion application]. C R    Acad Sci Hebd Seances Acad Sci D 262, 2543-5.-   Bystryak, S. (1998) U.S. Pat. No. 5,776,703. Immunoassay.-   Bystryak, S., Goldiner, I., Niv, A., Nasser, A. M. and    Goldstein, L. (1995) A homogeneous immunofluorescence assay based on    dye-sensitized photobleaching. Anal Biochem 225, 127-34.-   Catty, D. (1989) Antibodies: A Practical Approach, Vol. II. Oxford    University Press, Oxford. CDC. Bioterrorism Agents/Diseases. In,    Vol. 2013.-   Edberg, S. C. (1985) Principles of nucleic acid hybridization and    comparison with monoclonal antibody technology for the diagnosis of    infectious diseases. Yale J Biol Med 58, 425-42.-   Eglen, R. M., Reisine, T., Roby, P., Rouleau, N., Illy, C.,    Bosse, R. and Bielefeld, M. (2008) The use of AlphaScreen technology    in HTS: current status. Curr Chem Genomics 1, 2-10.-   Fleming, G. R., Knight, W. E. A., Morris, J. M., Morrison, R. J. S,    and Robinson, G. W. (1977) Picosecond fluorescence studies of    xanthene dyes. J Am Chem Soc 99, 4306-4311.-   Gandin, E., Lion, Y. and Van de Vorst, A. (1983) Quantum yield of    singlet oxygen production by xanthene derivatives. Photochem    Photobiol 37, 271-278.-   Ivnitski, D., Abdel-Hamid, I., Atanasov, P. and Wilkins, E. (1999)    Biosensors for detection of pathogenic bacteria. Biosensors &    Bioelectronics 14, 599-624.-   Jackson, P. and Blythe, D. (1993) Immunocytochemistry: Practical    Approach. Oxford University Press, Oxford.-   Mazenko, R. S., Rieders, F. and Brewster, J. D. (1999) Filtration    capture immunoassay for bacteria: optimization and potential for    urinalysis. J Microbiol Methods 36, 157-65.-   Meng, J. H., Zhao, S. H., Doyle, M. P. and Kresovich, S. (1996)    Polymerase chain-reaction for detecting E. coli 1157:H7. Intl J Food    Microbiol 32, 103-113.-   Motsenbocker, M., Masuya, H., Shimazu, H., Miyawaki, T.,    Ichimori, Y. and Sugawara, T. (1993a) Photoactive methylene blue dye    derivatives suitable for coupling to protein. Photochem Photobiol    58, 648-652.-   Motsenbocker, M., Sugawara, T., Shintani, M., Masuya, H.,    Ichimori, Y. and Kondo, K. (1993b) Establishment of the optically    pumped chemiluminescence technique for diagnostics. Anal Chem 65,    403-408.-   Nakane, P. K. and Pierce, G. B., Jr. (1966) Enzyme-labeled    antibodies: preparation and application for the localization of    antigens. J Histochem Cytochem 14, 929-31.-   Roche. (2003) AMPLICOR HIV-1-MONITOR test package insert. Roche    Diagnostic Systems, Branchburg, N.J.-   Sandell, J. H. and Masland, R. H. (1988) Photoconversion of some    fluorescent markers to a diaminobenzidine product. J Histochem    Cytochem 36, 555-9.-   Sawke, N. G. and Sawke, G. (2010) Preventing Post-Transfusion    Hepatitis by screening blood donors for IgM Antibody to Hepatitis B    core antigen. J Glob Infect Dis 2, 246-7.-   Schmidt, R. (2006a) Comment on “Quenching mechanism of rose bengal    triplet state involved in photosensitization of oxygen in ethylene    glycol”. Journal of Physical Chemistry A 110, 7749-7749.-   Schmidt, R. (2006b) Photosensitized generation of singlet oxygen.    Photochemistry and Photobiology 82, 1161-1177.-   Sperveslage, J., Stackebrandt, E., Lembke, F. W. and Koch, C. (1996)    Detection of bacterial contamination, including bacillus spores, in    dry growth media and in milk by identification of their 16S RDNA by    polymerase chain-reaction. J Microbiol Methods 26, 219-224.-   Timoshenko, V. (2009) SINGLET OXYGEN GENERATION AND DETECTION FOR    BIOMEDICAL APPLICATIONS. In: M.I. Baraton (Ed) Sensors for    Environment, Health and Security, Vol. II, NATO Science for Peace    and Security Series C p. 295-309.-   Ullman, E. F., Kirakossian, H., Singh, S., Wu, Z. P., Irvin, B. R.,    Pease, J. S., Switchenko, A. C., Irvine, J. D., Dafforn, A.,    Skold, C. N. and et al. (1994) Luminescent oxygen channeling    immunoassay: measurement of particle binding kinetics by    chemiluminescence. Proc Natl Acad Sci USA 91, 5426-30.-   Yu, H. and Bruno, J. G. (1996)    Immunomagnetic-electrochemiluminescent detection of Escherichia coli    O157 and Salmonella typhimurium in foods and environmental water    samples. Appl Environ Microbiol 62, 587-92.

What is claimed is:
 1. An assay for detecting an analyte in a fluidsample, the assay comprising the steps of: a) binding the analyte to afirst entity having an affinity to the analyte, said entity labeled witha photosensitizer or an enzyme to catalyze producing of saidphotosensitizer, b) adding to a mixture of said analyte and said entitya substrate for a photochemical reaction, said substrate is capable tobe converted to a product of the photochemical reaction when saidmixture of said entity, said analyte and said substrate is irradiatedwith a light at a wavelength within a light absorption spectrum of saidphotosensitizer, c) adding a reagent solution containing an additivedescribed by formula (1)

wherein said additive is an ascorbic acid or a derivative thereofobtained by at least one substitution at position 2, position 3,position 5, position 6 or any combinations thereof with at least onegroup of atoms selected from a group consisting of an anion group ofatoms, an aliphatic group of atoms not exceeding 16 carbon atoms, and anaromatic group of atoms not exceeding 16 carbon atoms; d) conducting thephotochemical reaction by irradiating the mixture of step (c) with saidlight, and e) detecting the analyte by measuring an optical signalcorresponding to the amount of the product of the photochemicalreaction.
 2. The assay as in claim 1, wherein said optical signal isselected from a group consisting of an optical density, reflectance,fluorescence, chemiluminescence and electrochemiluminescence of saidproduct of the photochemical reaction.
 3. The assay of claim 1, whereinthe photosensitizer is selected from a group consisting of a phenazine,a phenazine derivative, a 2,3-diamino-phenazine, an eosin, an eosinderivative, an erythrosine, an erythrosine derivative, a toluidine blue,a merocyanine 540, Rose Bengal, a methylene blue, a porphyrine, ahematoporphyrin, a porphyrine derivative, a phthalocyanine, aphthalocyanine derivative, an aluminum phthalocyanine tetrasulfonate(AlPCS) derivative, a riboflavin, and a quantum dot.
 4. The assay ofclaim 1, wherein the enzyme is selected from a group consisting of ahorseradish peroxidase, an alkaline phosphatase, a β-galactosidase, andtheir biotin-streptavidin complexes.
 5. The assay of claim 1, whereinthe substrate is selected from a group consisting of a phenylenediamine,a phenylenediamine derivative, a benzidine or a derivative thereof, adiaminobenzidine, 3,3′,5,5′-Tetramethylbenzidine, a olefin or aderivative thereof, a luminol or a derivative thereof, a dioxetane or aderivative thereof, a benzofurane or a derivative thereof,tyramine-biotin, and tyramine-photosensitizer.
 6. The assay as in claim1, wherein said substrate or said photosensitizer is embedded inmicroparticles, nanoparticles or liposomes.
 7. The assay of claim 1,wherein the analyte is present at a cell surface, a nitrocellulose or anylon membrane; the entity having an affinity to the analyte is anantibody against the analyte or a complex of primary and secondaryantibodies labeled with the photosensitizer or the enzyme to catalyzeproducing thereof, wherein the substrate is selected from a groupincluding a phenylenediamine derivative, a benzidine derivative, atyramine-biotin, and a tyramine-photosensitizer.
 8. The assay of claim1, wherein said step (a) further comprises adding a second entity withan affinity to the analyte, wherein said analyte binds to said firstentity and said second entity.
 9. The assay as in claim 8, wherein saidfirst entity is attached to a solid support embedding thephotosensitizer and said second entity is attached to a solid supportembedding the substrate.
 10. The assay of claim 8, wherein all steps arecarried out in a single mixture of all solutions without physicalseparation of bound and unbound fractions of all reagents.
 11. The assayof claim 8, wherein the assay is an ELISA-assay or a PCR-based assay,the first entity or the second entity is an antibody or aoligonucleotide, the enzyme to catalyze producing the photosensitizer isa horseradish peroxidase or biotin-streptavidin-horseradish peroxidasecomplex; the substrate is an orthophenylenediamine.
 12. The assay ofclaim 1, wherein the analyte is selected from a group consisting of aClostridium difficile toxin A, a Clostridium difficile toxin B, arotavirus, a Hepatitis B surface antigen, an HIV p24 antigen, a p50recombinant protein NFkB p50 homodimer, an RNA, a DNA, an mRNA, an cDNA,a prostatic specific antigen (PSA), an Anthrax (Bacillus anthracis), anArenaviruse, a Clostridium botulinum toxin, a Brucella specie, aBurkholderia pseudomallei, a Chlamydia psittaci, a Vibrio cholerae, anEbola virus, an Escherichia coli O157:H7, a variola major, aStaphylococcal enterotoxin B, a Francisella tularensis, a Salmonella, aRickettsia prowazekii, a Yersinia pestis, a Cryptosporidium parvum, andShigella.
 13. The assay of claim 1, wherein said additive in step (c) isan ascorbic acid or a derivative thereof obtained by at least onesubstitution at position 5, position 6 or both position 5 and position 6with at least one group of atoms selected from a group consisting of ananion group of atoms, an aliphatic group of atoms not exceeding 16carbon atoms, and an aromatic group of atoms not exceeding 16 carbonatoms.
 14. The assay of claim 1, wherein said additive in step (c) is anO-alkyl ascorbic acid derivative comprising said at least onesubstitution of R3 at position 5, R4 at position 6, or both R3 atposition 5 and R4 at position 6 by a group of (CH₂)x-CH₃, where x is notto exceed 14.